Anti-il-17a antibodies and their use in treating autoimmune and inflammatory disorders

ABSTRACT

The present disclosure relates to antibodies and proteins comprising an antigen-binding portion thereof that specifically bind to the pro-inflammatory cytokine IL-17 A. The disclosure more specifically relates to specific antibodies and proteins that are IL-17 A antagonists (inhibit the activities of IL-17 A and IL-17 AF) and are capable of inhibiting IL-17 A induced cytokine production in in vitro assays, and having an inhibitory effect in an antigen-induced arthritis model in vivo. The disclosure further relates to compositions and methods of use for said antibodies and proteins to treat pathological disorders that can be treated by inhibiting IL-17A or IL 17AF mediated activity, such as rheumatoid arthritis, psoriasis, systemic lupus erythematosus (SLE), lupus nephritis, chronic obstructive pulmonary disease, asthma or cystic fibrosis or other autoimmune and inflammatory disorders.

RELATED APPLICATIONS

The present disclosure is a divisional of U.S. patent application Ser.No. 15/906,446 filed Feb. 27, 2018 which is a divisional of U.S. patentapplication Ser. No. 15/466,128, filed Mar. 22, 2017, which is adivisional of U.S. patent application Ser. No. 14/859,872, filed Sep.21, 2015, which is a divisional of U.S. patent application Ser. No.14/174,942, filed Feb. 7, 2014 which claims priority to U.S. ProvisionalApplication No. 61/762,406, filed 8 Feb. 2013, which is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to antibodies and proteins comprising anantigen-binding portion thereof that specifically bind to IL-17A. Thedisclosure more specifically relates to specific antibodies and proteinsthat inhibit the effects of IL-17A and are capable of inhibitingIL-17A-induced activity, as well as compositions and methods of use forsaid antibodies and proteins, e.g. to treat pathological disorders thatcan be treated by inhibition of IL-17A signaling, for example autoimmuneand inflammatory disorders such as rheumatoid arthritis, psoriasis,systemic lupus erythematosus (SLE), lupus nephritis, multiple sclerosis,or chronic obstructive pulmonary disease, asthma or cystic fibrosis.

BACKGROUND OF THE INVENTION

Interleukin-17A (IL-17A also sometimes called IL-17) is the centrallymphokine produced by a newly defined subset of inflammatory T cells,the Th17 cells. In several animal models, these cells are pivotal forvarious autoimmune and inflammatory processes. Increased levels ofIL-17A have been associated with uveitis (Ambadi-Obi, et al 2007, NatureMed; 13:711-718), rheumatoid arthritis (RA), psoriasis, airwayinflammation, chronic obstructive pulmonary disease (COPD), inflammatorybowel disease (Crohn's disease and ulcerative colitis), allograftrejection, cancer, intra-peritoneal abscesses and adhesions, andmultiple sclerosis (Weaver, et al 2007, Annu Rev Immunol; 25:821-852;Witowski et al 2004, Cell Mol Life Sci; 61:567-579). Th17 cells canrapidly initiate an inflammatory response that is dominated byneutrophils (Miossec, et al 2009, NEJM; 361:888-98).

IL-17A was originally identified as a transcript from a rodent T-cellhybridoma. It is the founding member of a group of cytokines called theIL-17 family. Known as CTLA8 in rodents, IL-17A shows high homology toviral IL-17A encoded by an open reading frame of the T-lymphotropicrhadinovirus herpesvirus saimiri (Rouvier E, et al 1993, J. Immunol.150: 5445-56).

IL-17A is a cytokine that acts as a potent mediator in delayed-typereactions by increasing chemokine production in various tissues torecruit monocytes and neutrophils to the site of inflammation, similarto interferon gamma. The IL-17 family functions in the role ofproinflammatory cytokines that respond to the invasion of the immunesystem by extracellular pathogens and induces destruction of thepathogen's cellular matrix. IL-17A acts synergistically with tumornecrosis factor and interleukin-1 (Miossec P, et al 2009, N. Engl. J.Med. 361:888-98).

To elicit its functions, IL-17A binds to a type I cell surface receptorcalled IL-17R of which there are at least two variants, IL-17RA andIL-17RC (Pappu R, et al 2012, Trends Immunol.; 33:343-9). IL-17RA bindsIL-17A, IL-17AF and IL-17F and is expressed in multiple tissues:vascular endothelial cells, peripheral T cells, B cell lineages,fibroblast, lung, myelomonocytic cells, and marrow stromal cells (KollsJ K, Linden A 2004, Immunity 21:467-76; Kawaguchi M, et al 2004, J.Allergy Clin. Immunol. 114:1265-73; Moseley T A, et al 2003, CytokineGrowth Factor Rev. 14:155-74).

In addition to IL-17A, members of the IL-17 family include IL-17B,IL-17C, IL-17D, IL-17E (also called IL-25), and IL-17F. All members ofthe IL-17 family have a similar protein structure, with four highlyconserved cysteine residues critical to their 3-dimensional shape.Phylogenetic analysis reveals that among IL-17 family members, theIL-17F isoforms 1 and 2 (ML-1) have the highest homology to IL-17A(sharing 55 and 40% amino acid identity to IL-17A respectively),followed by IL-17B (29%), IL-17D (25%), IL-17C (23%), and IL-17E beingmost distantly related to IL-17A (17%). These cytokines are all wellconserved in mammals, with as much as 62-88% of amino acids conservedbetween the human and mouse homologs (Kolls J K, Linden A 2004, Immunity21:467-76). IL-17A is a 155-amino acid protein that is adisulfide-linked, homodimeric, secreted glycoprotein with a molecularmass of 35 kDa (Kolls J K, Lindén A 2004, Immunity 21:467-76). Thestructure of IL-17A consists of a signal peptide followed by the aminoacid region characteristic of the IL-17 family. An N-linkedglycosylation site on the protein was first identified afterpurification of the protein revealed two bands in standard SDS-PAGEanalysis, one at 15 kDa and another at 20 kDa. Comparison of differentmembers of the IL-17 family revealed four conserved cysteines that formtwo disulfide bonds (Yao Z, et al 1995, J. Immunol. 155:5483-6). IL-17is unique in that it bears no resemblance to other known interleukins.Furthermore, IL-17 bears no resemblance to any other known proteins orstructural domains (Kolls J K, Linden A2004, Immunity 21:467-76).Generally, other members of the IL-17 family such as IL-17F formhomodimers (like IL-17A).

IL-17A is also known to form a heterodimer with IL-17F under certaincircumstances. Heterodimeric IL-17AF is also produced by Th17 cellsfollowing stimulation by IL-23.

IL-17AF is thought to signal through the IL-17RA and IL-17RC receptorslike IL-17A and IL-17F. The biological functions of IL-17AF are similarto those of IL-17A and IL-17F. Stimulation of target cells by IL-17AFinduces the production of a variety of chemokines, in addition to airwayneutrophilia in appropriate circumstances. IL-17AF is considered to beless potent in these activities than homodimeric IL-17A, but more potentthan homodimeric IL-17F. For example, if the potency of IL-17A is 1,then the relative potency of IL-17AF is about 1/10 of that of IL-17A andthe relative potency of IL-17F is about 1/100 of that of IL-17A. Humanand mouse IL-17AF both show activity on mouse cells. IL-17AF consists ofa total of 271 amino acids and has a molecular weight of approximately30.7 kDa (data from product description of Human IL-17AF Heterodimerfrom Shenandoah Biotechnology).

A number of relevant crystal structures have been published. Theseinclude the crystal structure for homodimeric IL-17F (Hymowitz et al2001, EMBO J, 19:5332-5341).

The crystal structure of IL-17F in complex with the receptor IL-17RA hasalso been published (Ely et al., 2009 Nature Immunology 10:1245-1251).In addition at least one crystal structure of IL-17A in complex with theFab fragment of an antibody has been published (Gerhardt et al., 2009Journal of Molecular Biology, 5:905-921).

Several inflammatory and autoimmune diseases including psoriasis arelinked to exacerbated Th1 and/or Th17 responses. Many of them arecurrently treated either with general immunosuppressants or veryselectively acting biologicals such as anti-TNF-α antibodies that arenot effective in all patients. These were found to increase the risk forinfections and to become ineffective after repeated treatment.Therefore, there is an unmet medical need for treatments with increasedsafety profiles and simultaneous capacity to induce long-term remissionor cure of the disease.

Numerous immune regulatory functions have been reported for the IL-17family of cytokines, it is presumed due to their induction of manyimmune signaling molecules. The most notable role of IL-17A is itsinvolvement in inducing and mediating proinflammatory responses. IL-17Ais also associated with allergic responses. IL-17 induces the productionof many other cytokines (such as IL-6, G-CSF, GM-CSF, IL-1β, TGF-β,TNF-α), chemokines (including IL-8, GRO-α, and MCP-1), andprostaglandins (e.g., PGE2) from many cell types (fibroblasts,endothelial cells, epithelial cells, keratinocytes, and macrophages).The release of cytokines causes many functions, such as airwayremodeling, a characteristic of IL-17A responses. The increasedexpression of chemokines attracts other cells including neutrophils butnot eosinophils. IL-17 function is also essential to a subset of CD4+T-cells called T helper 17 (Th17) cells. As a result of these roles, theIL-17 family has been linked to many immune/autoimmune related diseasesincluding rheumatoid arthritis, asthma, lupus, allograft rejection andanti-tumor immunity (Aggarwal S, Gurney Ala. 2002, J. Leukoc. Biol.71:1-8). Additionally, links have been drawn to further conditions suchas osteoarthritis, septicemia, septic or endotoxic shock, allergicreactions, bone loss, psoriasis, ischemia, systemic sclerosis, fibrosis,and stroke.

Thus, there is a need for specific antibodies that antagonize theeffects of IL-17A and are capable of inhibiting IL-17A induced activity,and especially compositions and methods of use for said antibodies totreat pathological disorders that can be treated by inhibition of IL-17Asignaling.

SUMMARY OF THE INVENTION

Therefore, in one aspect, the disclosure provides an isolated antibodyor protein comprising an antigen-binding portion of an antibody,comprising CDR amino acid sequences having at least 95% identity tothose encoded by SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 3, and CDRamino acid sequence having at least 64% identity to those encoded by SEQID NO: 42, SEQ ID NO: 23 and SEQ ID NO: 11, and wherein said antibody ormolecule specifically binds to homodimeric IL-17A and heterodimericIL-17AF, but does not specifically bind to homodimeric IL-17F.

In one embodiment, the IL-17A, IL-17AF or IL-17F are selected from oneor more, such as two or three or more, of cynomolgus monkey, rhesusmacaque monkey, marmoset monkey, rat, mouse or human. In one specificembodiment, the IL-17A, IL-17AF or IL-17F is from human. In one specificembodiment, the IL-17A, IL-17AF or IL-17F is from human and mouse. Inone specific embodiment, the IL-17A, IL-17AF or IL-17F is fromcynomolgus monkey, rhesus macaque monkey, marmoset monkey, rat, mouseand human.

In one specific embodiment, the isolated antibody or protein comprisingan antigen-binding portion thereof of the disclosure, comprises an aminoacid sequence having at least 95% identity to SEQ ID NO: 12, and anamino acid sequence having at least 90% identity to SEQ ID NO: 43. Inone embodiment, the isolated antibody or comprises an amino acidsequence having at least 95% identity to SEQ ID NO: 14, and an aminoacid sequence having at least 95% identity to SEQ ID NO: 44.

In one embodiment, the isolated antibody or protein comprising anantigen-binding portion thereof comprises a light chain variable regioncomprising a CDR1, a CDR2, and a CDR3 domain selected from the groupconsisting of a) a light chain CDR1 domain of SEQ ID NO: 73, wherein thefirst variable amino acid is selected from the group consisting of Gly(G) and Val (V); the second variable amino acid is selected from thegroup consisting of Tyr (Y), Asn (N) and Ile (I); the third variableamino acid is selected from the group consisting of Trp (W) and Ser (S),and the fourth variable amino acid is selected from the group consistingof Glu (E) and Ala (A); b) a light chain CDR2 domain of SEQ ID NO: 74,wherein the variable amino acid is selected from the group consisting ofAsn (N) and Gln (Q); and c) a light chain CDR3 domain of SEQ ID NO: 75,wherein the variable amino acid is selected from the group consisting ofAsn (N) and Asp (D).

In one embodiment, the isolated antibody or protein comprising anantigen-binding portion thereof comprises heavy chain CDRs comprising,in sequence, a) SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 3, and lightchain CDRs comprising, in sequence, b) SEQ ID NO: 42, SEQ ID NO: 23 andSEQ ID NO: 11, c) SEQ ID NO: 42, SEQ ID NO: 10 and SEQ ID NO: 11, d) SEQID NO: 34, SEQ ID NO: 23 and SEQ ID NO: 11, e) SEQ ID NO: 22, SEQ ID NO:23 and SEQ ID NO: 24, or f) SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO:11.

In one specific embodiment, the isolated antibody or protein comprisingan antigen-binding portion thereof comprises heavy chain CDRs, insequence, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 3 and light chainCDRs, in sequence, SEQ ID NO: 42, SEQ ID NO: 23 and SEQ ID NO: 11.

In another specific embodiment, the isolated antibody or proteincomprising an antigen-binding portion thereof comprises heavy chainCDRs, in sequence, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 3 and lightchain CDRs, in sequence SEQ ID NO: 42, SEQ ID NO: 10 and SEQ ID NO: 11.

In another specific embodiment, the isolated antibody or proteincomprising an antigen-binding portion thereof comprises heavy chainCDRs, in sequence, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 3 and lightchain CDRs, in sequence, SEQ ID NO: 34, SEQ ID NO: 23 and SEQ ID NO: 11.

In another specific embodiment, the isolated antibody or proteincomprising an antigen-binding portion thereof comprises heavy chainCDRs, in sequence, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 3 and lightchain CDRs, in sequence, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24.

In another specific embodiment, the isolated antibody or proteincomprising an antigen-binding portion thereof comprises heavy chainCDRs, in sequence, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 3 and lightchain CDRs, in sequence, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.

In one embodiment, the isolated antibody or protein comprising anantigen-binding portion thereof comprises an immunoglobulin heavy chaincomprising a) SEQ ID NO: 12, and an immunoglobulin light chaincomprising b) SEQ ID NO: 43, c) SEQ ID NO: 53, d) SEQ ID NO: 35, e) SEQID NO: 25, or f) SEQ ID NO: 13.

In one specific embodiment, the isolated antibody or protein comprisingan antigen-binding portion thereof comprises an immunoglobulin heavychain according to SEQ ID NO: 12 and an immunoglobulin light chainaccording to SEQ ID NO: 43.

In one specific embodiment, the isolated antibody or protein comprisingan antigen-binding portion thereof comprises an immunoglobulin heavychain according to SEQ ID NO: 12 and an immunoglobulin light chainaccording to SEQ ID NO: 53.

In one specific embodiment, the isolated antibody or protein comprisingan antigen-binding portion thereof comprises an immunoglobulin heavychain according to SEQ ID NO: 12 and an immunoglobulin light chainaccording to SEQ ID NO: 35.

In one specific embodiment, the isolated antibody or protein comprisingan antigen-binding portion thereof comprises an immunoglobulin heavychain according to SEQ ID NO: 12 and an immunoglobulin light chainaccording to SEQ ID NO: 25.

In one specific embodiment, the isolated antibody or protein comprisingan antigen-binding portion thereof comprises an immunoglobulin heavychain according to SEQ ID NO: 12 and an immunoglobulin light chainaccording to SEQ ID NO: 13.

In one embodiment, the isolated antibody or protein comprising anantigen-binding portion thereof comprises an immunoglobulin heavy chaincomprising a) SEQ ID NO: 14, and an immunoglobulin light chaincomprising b) SEQ ID NO: 44, c) SEQ ID NO: 54, d) SEQ ID NO: 36, e) SEQID NO: 26, or f) SEQ ID NO: 15.

In one specific embodiment, the isolated antibody or protein comprisingan antigen-binding portion thereof comprises an immunoglobulin heavychain according to SEQ ID NO: 14, and an immunoglobulin light chainaccording to SEQ ID NO: 44.

In one specific embodiment, the isolated antibody or protein comprisingan antigen-binding portion thereof comprises an immunoglobulin heavychain according to SEQ ID NO: 14, and an immunoglobulin light chainaccording to SEQ ID NO: 54.

In one specific embodiment, the isolated antibody or protein comprisingan antigen-binding portion thereof comprises an immunoglobulin heavychain according to SEQ ID NO: 14, and an immunoglobulin light chainaccording to SEQ ID NO: 36.

In one specific embodiment, the isolated antibody or protein comprisingan antigen-binding portion thereof comprises an immunoglobulin heavychain according to SEQ ID NO: 14, and an immunoglobulin light chainaccording to SEQ ID NO: 26.

In one specific embodiment, the isolated antibody or protein comprisingan antigen-binding portion thereof comprises an immunoglobulin heavychain according to SEQ ID NO: 14, and an immunoglobulin light chainaccording to SEQ ID NO: 15.

In one aspect of the disclosure, an isolated antibody or proteincomprising an antigen-binding portion thereof is provided, which bindsto the same epitope as an isolated antibody or molecule according tospecific embodiments of the disclosure.

In one embodiment, the isolated antibody or protein comprising anantigen-binding portion thereof binds to an IL-17A epitope, such as ahuman IL-17A epitope, which comprises Arg78, Glu80, and Trp90.

The IL-17A epitope may further comprise Tyr85 or Arg124.

In one embodiment, the IL-17A epitope, such as a human IL-17A epitope,further comprises one or more of Pro82, Ser87 or Va188.

In one aspect of the disclosure, an isolated antibody or proteincomprising an antigen-binding portion thereof is provided, whichcomprises an antigen recognition surface having epitope recognitioncharacteristics equivalent to an antibody or molecule according tospecific embodiments.

In one aspect of the disclosure, an isolated antibody or proteincomprising an antigen-binding portion thereof is provided which iscross-blocked from binding to IL-17A, such as human IL-17A, or IL-17AF,such as human IL-17AF, by at least one antibody or protein comprising anantigen-binding portion thereof according to specific embodiments.

In one embodiment, the antibody or protein comprising an antigen-bindingportion thereof does not specifically bind to a) any one or more ofhuman IL-17F homodimer, IL-17B homodimer, IL-17C homodimer, IL-17Dhomodimer, IL-17E homodimer, and/or b) any one or more of cynomolgusmonkey IL-17F homodimer, mouse IL-17F homodimer, and/or c) any one ormore of other human cytokines selected from the group consisting ofIL-1, IL-3, IL-4, IL-6, IL-8, gIFN, TNF alpha, EGF, GMCSF, TGF beta 2,and/or d) any one or more of other mouse cytokines, selected from thegroup consisting of IL-1b, IL-2, IL-4, IL-6, IL-12, IL18, IL23, IFN orTNF.

In one embodiment, the antibody or protein comprising an antigen-bindingportion thereof binds to IL-17A, such as human IL-17A, so that theantibody or protein comprising an antigen-binding portion thereofinhibits or blocks binding between IL-17A and its receptor, and reducesor neutralizes IL-17A activity.

In one embodiment, the binding affinity of the antibody or proteincomprising an antigen-binding portion thereof for human IL-17A is 100 nMor less, 10 nM or less, 1 nM or less, 100 pM or less, or 10 pM or lessas measured by Biacore™. In a specific embodiment, the binding affinityof the antibody or protein comprising an antigen-binding portion thereoffor human IL-17A is below 200 pM, or below 100 pM, as measured byBiacore™.

In one embodiment, the antibody or protein comprising an antigen-bindingportion thereof is capable of inhibiting IL-6 secretion, or GRO-alphasecretion when assessed in vitro, preferably using cultured chondrocytesor fibroblasts.

In one embodiment, the antibody or protein comprising an antigen-bindingportion thereof is capable of inhibiting knee swelling in an antigeninduced arthritis experimental model in vivo, such as a rat AIA-model.

In one embodiment, the antibody or protein comprising an antigen-bindingportion thereof is conjugated to a further active moiety.

The antibody or protein comprising an antigen-binding portion thereofmay be monoclonal antibody or an antigen-binding portion thereof,preferably a chimeric, humanized, or human antibody or portion thereof.

In an aspect of the disclosure, a pharmaceutical composition isprovided, comprising an antibody or protein comprising anantigen-binding portion thereof according to embodiments of thedisclosure, in combination with one or more pharmaceutically acceptableexcipient, diluent or carrier.

In an embodiment, the pharmaceutical composition comprises one or moreadditional active ingredients.

In one specific embodiment, said pharmaceutical composition is alyophilisate. In another specific embodiment, the pharmaceuticalcomposition is a liquid formulation comprising a therapeuticallyacceptable amount of an antibody or molecule of the disclosure,preferably prepared as a pre-filled syringe.

The disclosure further relates to the use of said antibody or proteincomprising an antigen-binding portion thereof of the disclosure, inparticular XAB1, XAB2, XAB3, XAB4 or XAB5 antibodies, for use as amedicament, more preferably, for the treatment of a pathologicaldisorder that is mediated by IL-17A or that can be treated by inhibitionof IL-17A signaling, or IL-6 or GRO-alpha secretion.

In one specific embodiment, the antibodies or proteins comprising anantigen-binding portion thereof of the disclosure may be used for thetreatment of autoimmune and inflammatory disorders, such as arthritis,rheumatoid arthritis, psoriasis, chronic obstructive pulmonary disease,systemic lupus erythematosus (SLE), lupus nephritis, asthma, multiplesclerosis, or cystic fibrosis.

In one aspect of the disclosure, a use of said antibody or proteincomprising an antigen-binding portion thereof of the disclosure, inparticular XAB1, XAB2, XAB3, XAB4 or XAB5 antibodies, in the manufactureof a medicament for use in the treatment of a pathological disordermediated by IL-17A or that can be treated by inhibiting IL-6 orGRO-alpha secretion is provided.

In one specific embodiment, the a pathological disorder mediated byIL-17A or that can be treated by inhibiting IL-6 or GRO-alpha secretionis an inflammatory disorder or condition, such as arthritis, rheumatoidarthritis, psoriasis, chronic obstructive pulmonary disease, systemiclupus erythematosus (SLE), lupus nephritis, asthma, multiple sclerosisor cystic fibrosis.

In one aspect of the disclosure, a method of treating a pathologicaldisorder mediated by IL-17A, or that can be treated by inhibiting IL-6or GRO-alpha secretion is provided, said method comprising administeringan effective amount of an isolated antibody or molecule according to thedisclosure, in particular XAB1, XAB2, XAB3, XAB4 or XAB5 antibodies,such that the condition is alleviated.

In an embodiment, the condition is an inflammatory disorder orcondition, such as arthritis, rheumatoid arthritis, psoriasis, chronicobstructive pulmonary disease, systemic lupus erythematosus (SLE), lupusnephritis, asthma, multiple sclerosis or cystic fibrosis.

The disclosure also relates to the means for producing the antibodies orprotein comprising an antigen-binding portion thereof of the disclosure.Such means include isolated nucleic acid molecules encoding at least theheavy and/or light variable region(s) of the antibody or protein of thedisclosure or cloning expression vectors comprising such nucleic acids,in particular, for the recombinant production of an antibody or proteinaccording to the disclosure, for example XAB1, XAB2, XAB3, XAB4 or XAB5,in a host cell. In a specific embodiment, such cloning or expressionvector comprises at least one nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 18, 31, 51, 19, 28, 32, 38, 40, 46, 48,52, 56, and 58. In another embodiment, it comprises at least one of thefollowing coding sequences of variable heavy and light chain sequencesselected from the group consisting of SEQ ID NO: 16, 29, 49, 17, 27, 30,37, 39, 45, 47, 50, 55, and 57, operatively linked to suitable promotersequences, which are well known to a person skilled in the art.

In an embodiment, the nucleic acid molecule is a messenger RNA (mRNA),

The disclosure further relates to a host cell comprising one or morecloning or expression vectors as described above and to the process forthe production of an antibody or protein comprising an antigen-bindingportion thereof of the disclosure, in particular XAB1, XAB2, XAB3, XAB4or XAB5, said process comprising culturing the host cell, purifying andrecovering said antibody or protein.

Definitions

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

A “signal transduction pathway” or “signaling activity” refers to abiochemical causal relationship generally initiated by a protein-proteininteraction such as binding of a growth factor to a receptor, resultingin transmission of a signal from one portion of a cell to anotherportion of a cell. In general, the transmission involves specificphosphorylation of one or more tyrosine, serine, or threonine residueson one or more proteins in the series of reactions causing signaltransduction. Penultimate processes typically include nuclear events,resulting in a change in gene expression.

A naturally occurring “antibody” is a glycoprotein comprising at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as V_(H)) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, C_(L). The V_(H)and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system.

The terms “complementarity determining region,” and “CDR,” as usedherein refer to the sequences of amino acids within antibody variableregions which confer antigen specificity and binding affinity. Ingeneral, there are three CDRs in each heavy chain variable region(HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region(LCDR1, LCDR2, LCDR3).

The precise amino acid sequence boundaries of a given CDR can bedetermined using any of a number of well-known schemes, including thosedescribed by Kabat et al. (1991), “Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (“Kabat” numbering scheme),Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme).For example, for classic formats, under Kabat, the CDR amino acidresidues in the heavy chain variable domain (VH) are numbered 31-35(HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acidresidues in the light chain variable domain (VL) are numbered 24-34(LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia the CDR aminoacids in the VH are numbered 26-32 (HCDR1′), 52-56 (HCDR2′), and 95-102(HCDR3′); and the amino acid residues in VL are numbered 26-32 (LCDR1′),50-52 (LCDR2′), and 91-96 (LCDR3′). By combining the CDR definitions ofboth Kabat and Chothia, the CDRs consist of amino acid residues 26-35(HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acidresidues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.

The term “antigen-binding portion” of an antibody (or simply “antigenportion”), as used herein, refers to full length or one or morefragments of an antibody, such as a protein, that retain the ability tospecifically bind to an antigen (e.g., a portion of IL-17A). It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude a Fab fragment, a monovalent fragment consisting of the V_(L),V_(H), C_(L) and CH1 domains; a F(ab)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; a Fd fragment consisting of the V_(H) and CH1 domains; a Fvfragment consisting of the V_(L) and V_(H) domains of a single arm of anantibody; a dAb fragment (Ward et al. 1989, Nature 341:544-546), whichconsists of a V_(H) domain; and an isolated complementarity determiningregion (CDR), or any fusion proteins comprising such antigen-bindingportion.

Accordingly, the term “antigen-binding portion” may also refer to theportions corresponding to the antibody of the disclosure that may becomprised within alternative frameworks or scaffolds such as camelidantibodies or ‘non-antibody’ molecules as described below.

Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single chain protein in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al. 1988, Science 242:423-426; and Huston et al. 1988, Proc.Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. These antibody fragments are obtained using conventionaltechniques known to those of skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

An “isolated antibody”, as used herein, refers to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds toIL-17A, such as human IL-17A, is substantially free of antibodies thatspecifically bind to other antigens than IL-17A). An isolated antibodythat specifically binds to IL-17A may, however, have cross-reactivity toother antigens, such as IL-17A molecules from other species, or IL-17Aheterodimers, such as IL-17AF. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term IL-17A refers to human IL-17A as defined in SEQ ID NO: 76 orSEQ ID NO: 78 unless otherwise described. The term IL-17F refers tohuman IL-17F as defined in SEQ ID NO: 77 unless otherwise described.IL-17AF is a heterodimer of an IL-17A subunit and an IL-17F subunit, aswill be appreciated by a person skilled in the art. Recombinantproteins, designated with the prefix “r”, from different species wereused in the assays described below. For example, recombinant humanIL-17A is designated rhuIL-17. A person skilled in the art knows how toexpress such proteins using starting materials and standard protocols asknown in the art. However, in order to aid the skilled artisan, unlessotherwise stated, the following amino acid sequences may be used:cynomolgus monkey (cyno) IL-17A, SEQ ID NO: 79; cynoIL-17F, SEQ ID NO:80; rhesus monkey (rhesus) IL-17A, SEQ ID NO: 81; marmoset monkey(marmoset) IL-17A, SEQ ID NO: 82; mouse (m) IL-17A, SEQ ID NO: 83;mIL-17F, SEQ ID NO: 84, rat IL-17A, SEQ ID NO: 85; human IL-17 receptorA (huIL-17RA), SEQ ID NO: 86. As is known to a person skilled in theart, the abovementioned sequences may vary slightly, i.e. due tooriginating from different population groups. In the examples, toolantibodies are also used e.g. for screening purposes. Such antibodiesare standard antibodies and can be readily obtained by a person skilledin the art.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

The term “isotype” refers to the antibody class (e.g., IgM, IgE, IgGsuch as IgG1 or IgG4) that is provided by the heavy chain constantregion genes. Isotype also includes modified versions of one of theseclasses, where modifications have been made to alter the Fc function,for example, to enhance or reduce effector functions or binding to Fcreceptors.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from sequences of human origin. Furthermore, if theantibody contains a constant region, the constant region also is derivedfrom such human sequences, e.g., human germline sequences, or mutantversions of human germline sequences or antibody containing consensusframework sequences derived from human framework sequences analysis, forexample, as described in Knappik, et al. 2000, J Mol Biol 296:57-86).

A “humanized” antibody is an antibody that retains the reactivity of anon-human antibody while being less immunogenic in humans. This can beachieved, for instance, by retaining the non-human CDR regions andreplacing the remaining parts of the antibody with their humancounterparts (i.e., the constant region as well as the frameworkportions of the variable region). See, e.g., Morrison et al. 1984, Proc.Natl. Acad. Sci. USA, 81:6851-6855; Morrison and Oi, 1988, Adv.Immunol., 44:65-92; Verhoeyen et al. 1988, Science, 239:1534-1536;Padlan 1991, Molec. Immun., 28:489-498; and Padlan 1994, Molec. Immun.,31:169-217. Other examples of human engineering technology include, butare not limited to Xoma technology disclosed in U.S. Pat. No. 5,766,886.

The human antibodies of the disclosure may include amino acid residuesnot encoded by human sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human sequences.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the human antibody, e.g., from atransfectoma, antibodies isolated from a recombinant, combinatorialhuman antibody library, and antibodies prepared, expressed, created orisolated by any other means that involve splicing of all or a portion ofa human immunoglobulin gene sequence to other DNA sequences. Suchrecombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM, IgE,IgG, such as IgG1 or IgG4) that is provided by the heavy chain constantregion genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen”.

As used herein, an antibody or a protein that “specifically binds toIL-17A polypeptide” is intended to refer to an antibody or protein thatbinds to human IL-17A polypeptide with a K_(D) of 100 nM or less, 10 nMor less, 1 nM or less, 100 pM or less, or 10 pM or less. An antibodythat “cross-reacts with an antigen other than IL-17A” is intended torefer to an antibody that binds that antigen with a K_(D) of 10 nM orless, 1 nM or less, or 100 pM or less. An antibody that “does notcross-react with a particular antigen” is intended to refer to anantibody that binds to that antigen, with a K_(D) of 100 nM or greater,or a K_(D) of 1 μM or grater, or a K_(D) of 10 μM or greater. In certainembodiments, such antibodies that do not cross-react with the antigenexhibit essentially undetectable binding against these proteins instandard binding assays.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction.

The term “K_(D)”, as used herein, is intended to refer to thedissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e. K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A method for determining the K_(D) of anantibody is by using surface plasmon resonance, or using a biosensorsystem such as a Biacore™ system, well known to a person skilled in theart and operated e.g. as described in the Examples.

The inhibition of the binding of IL-17 to its receptor may beconveniently tested in various assays including such assays aredescribed hereinafter in the text. By the term “to the same extent” ismeant that the reference and the equivalent molecules exhibit, on astatistical basis, essentially identical IL-17 inhibitory activity inone of the assays referred to herein (see Examples). For example, IL-17binding molecules of the disclosure typically have a half maximalinhibitory concentration (IC₅₀), for the inhibition of human IL-17 onIL-6 production induced by human IL-17 in human dermal fibroblasts,which is within +/−10⁵, i.e. below 10 nM, more preferably 9, 8, 7, 6, 5,4, 3 or 2 nM of that of, preferably substantially the same as, the IC₅₀of the respective reference molecule when assayed e.g. as described inthe Examples. Alternatively, the assay used may be an assay ofcompetitive inhibition of binding of IL-17 by soluble IL-17 receptorsand the IL-17 binding molecules of the disclosure.

As used herein, the term “Affinity” refers to the strength ofinteraction between antibody and antigen at single antigenic sites.Within each antigenic site, the variable region of the antibody “arm”interacts through weak non-covalent forces with the antigen at numeroussites; the more interactions, the stronger the affinity. As used herein,the term “high affinity” for an IgG antibody or fragment thereof (e.g.,a Fab fragment) refers to an antibody having a K_(D) of 10⁻⁸ M or less,10⁻⁹ M or less, or 10⁻¹⁰ M, or 10⁻¹¹ M or less, or 10⁻¹² M or less, or10⁻¹³ M or less for a target antigen. However, high affinity binding can10 vary for other antibody isotypes. For example, high affinity bindingfor an IgM isotype refers to an antibody having a K_(D) of 10⁻⁷ M orless, or 10⁻⁸ M or less.

As used herein, the term “Avidity” refers to an informative measure ofthe overall stability or strength of the antibody-antigen complex. It iscontrolled by three major factors: antibody epitope affinity; thevalence of both the antigen and antibody; and the structural arrangementof the interacting parts. Ultimately these factors define thespecificity of the antibody, that is, the likelihood that the particularantibody is binding to a precise antigen epitope.

As used herein, an antibody or protein that inhibits IL-17A binding toIL-17R is intended to refer to an antibody or protein that inhibitsIL-17A binding to IL-17R with an IC₅₀ of 10 nM or less, preferably withan IC₅₀ of 1 nM or less, more preferably with an IC₅₀ of 100 pM, orless, as measured in an in vitro competitive binding assay. Such assayis described in more details in the examples below.

As used herein, the term “IL-17A antagonist” or “IL-17A blockingmolecule” is intended to refer to an antibody or protein that inhibitsIL-17A induced signaling activity through the IL-17R and thereby reducesor neutralizes IL-17A activity. This can be shown in a human cell assaysuch as the IL-17A dependent IL-6 or GRO-alpha production assay in humancells. Such assay is described in more detail in the Examples below. Insome embodiments, the antibodies or proteins of the disclosure inhibitIL-17A dependent IL-6 or GRO-alpha production as measured in an in vitrohuman cell assay at an IC₅₀ of 10 nM or less, 1 nM or less, or 100 pM orless. Such an assay is described in more details in the Examples below.In some embodiments the antibodies or proteins of the disclosure inhibitantigen induced arthritis in in vivo assays in mice and rats. Suchassays are described in the Examples in more detail below.

As used herein, the term “ADCC” or “antibody dependent cellcytotoxicity” activity refers to cell depleting activity. ADCC activitycan be measured by standard ADCC assay, well known to a person skilledin the art.

As used herein, the term “selectivity” for an antibody or protein of thedisclosure refers to an antibody or protein that binds to a certaintarget polypeptide, but not to closely related polypeptides. The phrases“an antibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich binds specifically to an antigen”.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. (See, U.S. Pat. No. 8,278,036 to Kariko et al.,which discloses mRNA molecules with uridine replaced by pseudouridine,methods of synthesizing the same, and methods for the delivery oftherapeutic proteins in vivo.) Methods for packaging mRNA can be used,for example, those disclosed in U.S. Pat. No. 8,278,036 to Kariko et al;and patent application WO2013/090186A1, to Moderna. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions), alleles, orthologs, SNPs, and complementarysequences as well as the sequence explicitly indicated. Specifically,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.,Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98(1994)).

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows, chickens, amphibians, reptiles, etc. As used herein, theterms “cyno” or “cynomolgus” refer to the cynomolgus monkey (Macacafascicularis). As used herein, the terms “rhesus” or “rhesus macaque”refer to the rhesus macaque monkey (Macaca mulatta). As used herein, theterm “marmoset” refers to a marmoset monkey.

As used herein, the term “treating” or “treatment” of any disease ordisorder (i.e., rheumatoid arthritis) refers in one embodiment, toameliorating the disease or disorder (i.e., slowing or arresting orreducing the development of the disease or at least one of the clinicalsymptoms thereof). In another embodiment “treating” or “treatment”refers to alleviating or ameliorating at least one physical parameterincluding those which may not be discernible by the patient. In yetanother embodiment, “treating” or “treatment” refers to modulating thedisease or disorder, either physically, (e.g., stabilization of adiscernible symptom), physiologically, (e.g., stabilization of aphysical parameter), or both. Methods for assessing treatment and/orprevention of disease are generally known in the art, unlessspecifically described herein.

As used herein, “selecting” and “selected” in reference to a patient isused to mean that a particular patient is specifically chosen from alarger group of patients due to the particular patient having apredetermined criterion. Similarly, “selectively treating a patient”refers to providing treatment to a patient that is specifically chosenfrom a larger group of patients due to the particular patient having apredetermined criteria. Similarly, “selectively administering” refers toadministering a drug to a patient that is specifically chosen from alarger group of patients due to the particular patient having apredetermined criterion.

As used herein, the term, “optimized” means that a nucleotide sequencehas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a cell of Pichia, a cell of Trichoderma, a ChineseHamster Ovary cell (CHO) or a human cell. The optimized nucleotidesequence is engineered to retain completely or as much as possible theamino acid sequence originally encoded by the starting nucleotidesequence, which is also known as the “parental” sequence. The optimizedsequences herein have been engineered to have codons that are preferredin CHO mammalian cells, however optimized expression of these sequencesin other eukaryotic cells is also envisioned herein. The amino acidsequences encoded by optimized nucleotide sequences are also referred toas optimized.

As used herein, the percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % identity=# of identical positions/total # of positions×100),taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm, as described below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller 1988, Comput. Appl.Biosci., 4:11-17, which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. Alternatively, the percent identitybetween two amino acid sequences can be determined using the Needlemanand Wunsch 1970, J. Mol, Biol. 48:444-453 algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The percent identity between two nucleotide amino acid sequences mayalso be determined using for example algorithms such as the BLASTNprogram for nucleic acid sequences using as defaults a word length (W)of 11, an expectation (E) of 10, M=5, N=4, and a comparison of bothstrands.

The terms “cross-block”, “cross-blocked”, “cross-blocking” are usedinterchangeably herein to mean the ability of an antibody or otherbinding agent to interfere with the binding of other antibodies orbinding agents to IL-17A in a standard competitive binding assay.

The ability or extent to which an antibody or other binding agent, suchas a protein comprising an antigen-binding portion of an antibody, isable to interfere with the binding of another antibody or bindingmolecule to IL-17A, and therefore whether it can be said to cross-blockaccording to the disclosure, can be determined using standardcompetition binding assays. One suitable assay involves the use of theBiacore™ technology (e.g. by using the Biacore™ 3000 instrument(Biacore™, Uppsala, Sweden)), which can measure the extent ofinteractions using surface plasmon resonance technology. Another assayfor measuring cross-blocking uses an ELISA-based approach. Furtherdetails on these methods are given in the Examples.

For example, the antibodies exemplified herein (i.e. XAB1, XAB2, XAB3,XAB4 and XAB5) and proteins comprising an antigen-binding portionthereof will all “cross-block” one another. All of these antibodiestarget the same epitope on IL-17A. Other cross-blocking antibodies wouldbe anticipated to bind to the same, or a related, epitope.

According to the disclosure, a cross-blocking antibody or other bindingagent, such as a protein comprising an antigen-binding portion of anantibody, according to the disclosure binds to IL-17A in the describedBiacore™ cross-blocking assay such that the recorded binding of thecombination (mixture) of the antibodies or binding agents is between 80%and 0.1% (e.g. 80% to 4%) of the maximum theoretical binding,specifically between 75% and 0.1% (e.g. 75% to 4%) of the maximumtheoretical binding, and more specifically between 70% and 0.1% (e.g.70% to 4%), and more specifically between 65% and 0.1% (e.g. 65% to 4%)of maximum theoretical binding (as defined above) of the two antibodiesor binding agents in combination

An antibody is defined as cross-blocking in the ELISA assay as describedin the Examples, if the solution phase anti-IL-17A antibody is able tocause a reduction of between 60% and 100%, specifically between 70% and100%, and more specifically between 80% and 100%, of the IL-17Adetection signal (i.e. the amount IL-17A bound by the coated antibody)as compared to the IL-17A detection signal obtained in the absence ofthe solution phase anti-IL-17A antibody (i.e. the positive controlwells).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, in part, on the discovery of antibodymolecules that specifically bind to homodimeric IL-17A and heterodimericIL-17AF, but do not specifically bind to homodimeric IL-17F. Thedisclosure relates to both full IgG format antibodies as well asproteins comprising an antigen-binding portion thereof, which will befurther described below.

Accordingly, the present disclosure provides antibodies as well asproteins comprising an antigen-binding portion thereof with bindingcapabilities that are surprisingly similar for several species, such asselected from one or more of cynomolgus, rhesus macaque, marmoset, rat,mouse or human, as well as pharmaceutical compositions, productionmethods, and methods of use of such antibodies and compositions.

Recombinant Antibodies

Antibodies of the disclosure include the human recombinant antibody XAB1and antibody derivates XAB2, XAB3, XAB4 and XAB5, which were derived,isolated and structurally characterized by their full length heavy andlight chain amino acid sequences as described in Table 1 below.

TABLE 1 Full length heavy and light chain amino acid sequences of XAB1,XAB2, XAB3, XAB4 and XAB5. Full Length Heavy Chain Full Length LightChain Antibody Amino acid sequence Amino acid sequence XAB1 SEQ ID NO:14 SEQ ID NO: 15 XAB2 SEQ ID NO: 14 SEQ ID NO: 26 XAB3 SEQ ID NO: 14 SEQID NO: 36 XAB4 SEQ ID NO: 14 SEQ ID NO: 44 XAB5 SEQ ID NO: 14 SEQ ID NO:54 SEQUENCE 100% 97% IDENTITY

The corresponding variable regions, V_(H) and V_(L) amino acid sequencesof such isolated antibodies XAB1, XAB2, XAB3, XAB4 and XAB5 of thedisclosure are shown in Table 2 below.

TABLE 2 Variable heavy and light chain amino acid sequences of XAB1,XAB2, XAB3, XAB4 and XAB5. Variable Heavy Chain Variable Light ChainAntibody Amino acid sequence Amino acid sequence XAB1 SEQ ID NO: 12 SEQID NO: 13 XAB2 SEQ ID NO: 12 SEQ ID NO: 25 XAB3 SEQ ID NO: 12 SEQ ID NO:35 XAB4 SEQ ID NO: 12 SEQ ID NO: 43 XAB5 SEQ ID NO: 12 SEQ ID NO: 53SEQUENCE 100% 94% IDENTITY

Other antibodies of the disclosure include those having amino acids thathave been mutated by amino acid deletion, insertion or substitution, yethave at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,98%, 99% or 100% identity to the antibodies described above, inparticular in the CDR regions depicted in the sequences described above.In some embodiments, the antibody of the disclosure is a mutant variantof any one of XAB1, XAB2, XAB3, XAB4 and XAB5, wherein said mutantvariant antibody include mutant amino acid sequences wherein no morethan 1, 2, 3, 4 or 5 amino acids have been mutated by amino aciddeletion, insertion or substitution in the CDR regions when comparedwith the CDR regions depicted in the sequences described above.

Full length light and heavy chains nucleotide coding sequences of XAB1,XAB2, XAB3, XAB4 and XAB5 are shown in Table 3 below.

TABLE 3 Full length heavy and light chain DNA coding sequences. FullLength Heavy Chain Full Length Light Chain Antibody DNA coding sequenceDNA coding sequence XAB1 SEQ ID NO: 18, SEQ ID NO: 19 SEQ ID NO: 31, SEQID NO: 51 XAB2 SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO:32 SEQ ID NO: 51 XAB3 SEQ ID NO: 18, SEQ ID NO: 38, SEQ ID NO: 31, SEQID NO: 40 SEQ ID NO: 51 XAB4 SEQ ID NO: 18, SEQ ID NO: 46, SEQ ID NO:31, SEQ ID NO: 48, SEQ ID NO: 51 SEQ ID NO: 52 XAB5 SEQ ID NO: 18, SEQID NO: 56, SEQ ID NO: 31, SEQ ID NO: 58 SEQ ID NO: 51

Variable light and heavy chains nucleotide coding sequences of XAB1,XAB2, XAB3, XAB4 and XAB5 are shown in Table 4 below.

TABLE 4 Variable heavy and light chain amino acid sequences DNA codingsequences. Variable Heavy Chain Variable Light Chain Antibody DNA codingsequence DNA coding sequence XAB1 SEQ ID NO: 16, SEQ ID NO: 17 SEQ IDNO: 29, SEQ ID NO: 49 XAB2 SEQ ID NO: 16, SEQ ID NO: 27, SEQ ID NO: 29,SEQ ID NO: 30 SEQ ID NO: 49 XAB3 SEQ ID NO: 16, SEQ ID NO: 37, SEQ IDNO: 29, SEQ ID NO: 39 SEQ ID NO: 49 XAB4 SEQ ID NO: 16, SEQ ID NO: 45,SEQ ID NO: 29, SEQ ID NO: 47, SEQ ID NO: 49 SEQ ID NO: 50 XAB5 SEQ IDNO: 16, SEQ ID NO: 55, SEQ ID NO: 29, SEQ ID NO: 57 SEQ ID NO: 49

Other nucleic acids encoding antibodies of the disclosure includenucleic acids that have been mutated by nucleotide deletion, insertionor substitution, yet have at least 60, 70, 80, 90, 95 or 100 percentidentity to the CDR corresponding coding regions depicted in thesequences described above or in Table 5 and Table 6 below.

In some embodiments, it include variant nucleic acids wherein no morethan 1, 2, 3, 4 or 5 nucleotides have been changed by nucleotidedeletion, insertion or substitution in the CDR coding regions with theCDR coding regions depicted in the sequences described above or in Table5 and Table 6 below.

For antibodies that bind to the same epitope, the V_(H), V_(L), fulllength light chain, and full length heavy chain sequences (nucleotidesequences and amino acid sequences) can be “mixed and matched” to createother anti-IL-17A binding molecules of the disclosure. IL-17A binding ofsuch “mixed and matched” antibodies can be tested using the bindingassays described above or other conventional binding assays (e.g.,ELISAs). When these chains are mixed and matched, a V_(H) sequence froma particular V_(H)/V_(L) pairing should be replaced with a structurallysimilar V_(H) sequence. Likewise a full length heavy chain sequence froma particular full length heavy chain/full length light chain pairingshould be replaced with a structurally similar full length heavy chainsequence. Likewise, a V_(L) sequence from a particular V_(H)/V_(L)pairing should be replaced with a structurally similar V_(L) sequence.Likewise a full length light chain sequence from a particular fulllength heavy chain/full length light chain pairing should be replacedwith a structurally similar full length light chain sequence.Accordingly, in one aspect, the disclosure provides an isolatedrecombinant antibody or protein comprising an antigen-binding portionthereof having: a heavy chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 12 and a lightchain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 13, 25, 35, 43 and 53; wherein saidheavy and light chain regions are selected such that the antibodyspecifically binds to IL-17A.

Examples of the amino acid sequences of the V_(H) CDR1s (also calledHCDR1 or HCDR1′ depending of the CDR definition that is used), V_(H)CDR2s (also called HCDR2 or HCDR2′ depending of the CDR definition thatis used), V_(H) CDR3s (also called HCDR1 or HCDR1′ depending of the CDRdefinition that is used), V_(L) CDR1s (also called LCDR1 or LCDR1′depending of the CDR definition that is used), V_(L) CDR2s (also calledLCDR2 or LCDR2′ depending of the CDR definition that is used), V_(L)CDR3s (also called HCDR3 or HCDR3′ depending of the CDR definition thatis used) of some antibodies and proteins comprising an antigen-bindingportion thereof according to the disclosure are shown in Table 5 andTable 6.

In Table 5, the CDR regions of some antibodies of the disclosure aredelineated using the Kabat system (Kabat, E. A., et al. 1991, Sequencesof Proteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, see also Zhao&Lu2009, Molecular Immunology 47:694-700)

For the ease of reading, when CDR regions are delineated according toKabat definition, they are called hereafter HCDR1, HCDR2, HCDR3, LCDR1,LCDR2, LCDR3 respectively.

TABLE 5 CDR regions of XAB1, XAB2, XAB3, XAB4 and XAB5 according toKabat definition. Original antibody HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3XAB1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 7 NO: 8 NO: 3 NO: 9NO: 10 NO: 11 XAB2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 7 NO: 8NO: 3 NO: 22 NO: 23 NO: 24 XAB3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQID NO: 7 NO: 8 NO: 3 NO: 34 NO: 23 NO: 11 XAB4 SEQ ID SEQ ID SEQ ID SEQID SEQ ID SEQ ID NO: 7 NO: 8 NO: 3 NO: 42 NO: 23 NO: 11 XAB5 SEQ ID SEQID SEQ ID SEQ ID SEQ ID SEQ ID NO: 7 NO: 8 NO: 3 NO: 42 NO: 10 NO: 11CONSENSUS SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 7 NO: 8 NO: 3NO: 73 NO: 74 NO: 75 SEQUENCE 100% 100% 100% 64% 86% 89% IDENTITY

The consensus sequences SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75comprise a number of variable amino acids, designated X. Based onsequence alignment of the sequences for XAB2 to XAB5, the four variableamino acids in SEQ ID NO: 73 can advantageously be selected according tothe following: The first variable amino acid (X1) can be selected fromthe group consisting of Gly (G) and Val (V); the second variable aminoacid (X2) can be selected from the group consisting of Tyr (Y), Asn (N)and Ile (I); the third variable amino acid (X3) can be selected from thegroup consisting of Trp (W) and Ser (S); and the fourth variable aminoacid (X4) can be selected from the group consisting of Glu (E) and Ala(A). SEQ ID NO: 9 has a sequence identity of 91% compared to SEQ ID NO:22 and a sequence identity of 73% compared to SEQ ID NO: 34 and SEQ IDNO: 42. SEQ ID NO: 22 has a sequence identity of 64% compared to SEQ IDNO: 34 and SEQ ID NO: 42. SEQ ID NO: 34 has a sequence identity of 91%compared to SEQ ID NO: 42.

Similarly, the one variable amino acid in SEQ ID NO: 74 canadvantageously be selected according to the following: X1 can beselected from the group consisting of Asn (N) and Gln (Q). SEQ ID NO: 10has a sequence identity of 86% compared to SEQ ID NO: 23.

The one variable amino acid in SEQ ID NO: 75 can advantageously beselected according to the following: X1 can be selected from the groupconsisting of Asn (N) and Asp (D). SEQ ID NO: 11 has a sequence identityof 89% compared to SEQ ID NO: 24.

In Table 6, the CDR regions of some antibodies of the disclosure aredelineated using the Chothia system, Al-Lazikani et al. 1997, J. Mol.Biol. 273:927-948. For ease of reading, when the CDR regions aredelineated according to Chothia definition, they are called hereafterHCDR1′, HCDR2′, HCDR3′, LCDR1′, LCDR2′, LCDR3′ respectively.

TABLE 6 CDR regions from XAB1, XAB2, XAB3, XAB4 and XAB5 according toChothia definition. Original antibody HCDR1′ HCDR2′ HCDR3′ LCDR1′ LCDR2′LCDR3′ XAB1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 2 NO: 3NO: 4 NO: 5 NO: 6 XAB2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 1NO: 2 NO: 3 NO: 20 NO: 5 NO: 21 XAB3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID NO: 1 NO: 2 NO: 3 NO: 33 NO: 5 NO: 6 XAB4 SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID NO: 1 NO: 2 NO: 3 NO: 41 NO: 5 NO: 6 XAB5 SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 2 NO: 3 NO: 41 NO: 5 NO: 6CONSENSUS SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 2 NO: 3NO: 71 NO: 5 NO: 72 SEQUENCE 100% 100% 100% 43% 100% 83% IDENTITY

The consensus sequences SEQ ID NO: 71 and SEQ ID NO: 72 comprise anumber of variable amino acids, designated X. Based on sequencealignment of the sequences for XAB2 to XAB5, the four variable aminoacids in SEQ ID NO: 71 can advantageously be selected according to thefollowing: the first variable amino acid (X1) can be selected from thegroup consisting of Gly (G) and Val (V), the second variable amino acid(X2) can be selected from the group consisting of Tyr (Y), Asn (N) andIle (I); the third variable amino acid (X3) can be selected from thegroup consisting of Trp (W) and Ser (5), and the fourth variable aminoacid (X4) can be selected from the group consisting of Glu (E) and Ala(A). SEQ ID NO: 4 has a sequence identity of 86% compared to SEQ ID NO:20 and a sequence identity of 57% compared to SEQ ID NO: 33 and SEQ IDNO: 41. SEQ ID NO: 20 has a sequence identity of 43% compared to SEQ IDNO: 33 and SEQ ID NO: 41. SEQ ID NO: 33 has a sequence identity of 86%compared to SEQ ID NO: 41.

Similarly, the one variable amino acid in SEQ ID NO: 72 canadvantageously be selected according to the following: X1 can beselected from the group consisting of Asn (N) and Asp (D). SEQ ID NO: 6has a sequence identity of 86% compared to SEQ ID NO: 21.

Given that each of these antibodies can bind to IL-17A and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the V_(H) CDR1, 2 and 3 sequences and V_(L) CDR1, 2 and 3sequences can be “mixed and matched” (i.e., CDRs from differentantibodies can be mixed and matched, each antibody containing a V_(H)CDR1, 2 and 3 and a V_(L) CDR1, 2 and 3 create other anti-IL-17A bindingmolecules of the disclosure). IL-17A binding of such “mixed and matched”antibodies can be tested using the binding assays described above and inthe Examples or other conventional assays (e.g., ELISAs). When V_(H) CDRsequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequencefrom a particular V_(H) sequence should be replaced with a structurallysimilar CDR sequence(s). Likewise, when V_(L) CDR sequences are mixedand matched, the CDR1, CDR2 and/or CDR3 sequence from a particular V_(L)sequence should be replaced with a structurally similar CDR sequence(s).It will be readily apparent to the ordinarily skilled artisan that novelV_(H) and V_(L) sequences can be created by substituting one or moreV_(H) and/or V_(L) CDR region(s) sequence(s) with structurally similarsequences from the CDR sequences shown herein for monoclonal antibodiesof the present disclosure.

In one embodiment, an isolated recombinant antibody, or a proteincomprising an antigen-binding portion thereof, has: a heavy chainvariable region CDR1 according to SEQ ID NO: 7; a heavy chain variableregion CDR2 according to SEQ ID NO: 8; a heavy chain variable regionCDR3 according to SEQ ID NO: 3; a light chain variable region CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 9, 22, 34, 42, and 73, and preferably selected from the groupconsisting of selected from the group consisting of SEQ ID NO: 9, 22,34, 42; a light chain variable region CDR2 comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 10, 23, and74, and preferably selected from the group consisting of selected fromthe group consisting of SEQ ID NO: 10 and 23; and a light chain variableregion CDR3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 11, 24, and 75 and preferably selected from thegroup consisting of selected from the group consisting of SEQ ID NO: 11and 24; wherein said CDR regions are selected so that the antibody orprotein of the disclosure specifically binds to IL-17A.

In another embodiment, an isolated recombinant antibody, or a proteincomprising an antigen-binding portion thereof has: a heavy chainvariable region HCDR1′ according to SEQ ID NO: 1; a heavy chain variableregion HCDR2′ according to SEQ ID NO: 2; a heavy chain variable regionHCDR3′ according to SEQ ID NO: 3; a light chain variable region LCDR1′comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 4, 20, 33, 41, and 71, and preferably selected from the groupconsisting of selected from the group consisting of SEQ ID NO: 4, 20,33, 41; a light chain variable region LCDR2′ according to SEQ ID NO: 5;and a light chain variable region LCDR3′ comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 6, 21, and 72,and preferably selected from the group consisting of selected from thegroup consisting of SEQ ID NO: 6 and 21; wherein said CDR regions areselected so that the antibody or protein of the disclosure specificallybinds to IL-17A.

In certain embodiments, the antibody or protein comprising anantigen-binding portion thereof comprises either SEQ ID NO: 7, SEQ IDNO: 8 and SEQ ID NO: 3; SEQ ID NO: 12; or c) SEQ ID NO: 14.

As used herein, a human antibody comprises heavy or light chain variableregions or full length heavy or light chains that are “the product of”or “derived from” a particular germline sequence if the variable regionsor full length chains of the antibody are obtained from a system thatuses human germline immunoglobulin genes. Such systems includeimmunizing a transgenic mouse carrying human immunoglobulin genes withthe antigen of interest or screening a human immunoglobulin gene librarydisplayed on phage with the antigen of interest. A human antibody thatis “the product of” or “derived from” a human germline immunoglobulinsequence can be identified as such by comparing the amino acid sequenceof the human antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody. A human antibody that is “the product of” or“derived from” a particular human germline immunoglobulin sequence maycontain amino acid differences as compared to the germline sequence, dueto, for example, naturally occurring somatic mutations or intentionalintroduction of site-directed mutation. However, a selected humanantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a human germline immunoglobulin geneand contains amino acid residues that identify the human antibody asbeing human when compared to the germline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a human antibody may be at least 60%, 70%, 80%, 90%, or at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

In the present disclosure, an epitope on IL-17A has been identified thatis particularly preferred as a target for the binding of potentiallytherapeutic antibodies. This epitope is bound by XAB1, and the variantantibodies XAB2, XAB3, XAB4 and XAB5 which have been developed bymodification of the sequence of XAB1. This epitope is found on theIL-17A sequence, between residues Arg 78 and Trp 90.

The epitope may be considered to comprise the following most preferredamino acid residues from within the IL-17A: Arg 78, Glu 80, Trp 90. Inaddition, the following amino acid residues are also preferred: Tyr 85,Arg 124. Other important amino acid residues are Pro 82, Ser 87, Val 88.Further contributing amino acid residues are Val 45*, Leu 49, Asp 81,Glu 83, Pro 86, Pro 130, Phe 133, Lys 137*, where amino acids markedwith (*) designate residue contributed by the second IL-17A subunit ofthe homodimer IL-17A.

Antibodies that target this epitope on IL-17A have been shown to blockthe binding of IL-17A to its receptor to inhibit IL-17A mediated effectsin vitro, and to reduce the severity of an experimental in vivo model ofantigen induced arthritis. In addition, antibodies that bind to thisepitope have unexpectedly been shown to inhibit the in vitro effectsmediated by the IL-17AF heterodimer, and also to retain an unexpectedlyhigh affinity for IL-17A and IL-17AF derived from mouse and otherspecies variations of the target molecule.

Thus, this epitope is especially preferred since it is also unexpectedlypreserved in an accessible format within the structure of the IL-17AFheterodimer. Accordingly, preferred antibodies of the disclosure willalso bind to IL-17AF heterodimers. Without wishing to be bound bytheory, it is anticipated that the structure of the IL-17AF heterodimeris sufficiently similar, to IL-17A, or that interaction with theantibodies of the disclosure renders it sufficiently similar to IL-17A,that binding may still occur.

This is unexpected because structural analyses based on the availablestructures for IL-17A, IL-17F and interactions between these moleculesand antibodies or receptors that have been obtained by X-raycrystallography (published in the art and conducted by the inventors) incombination with in silico predictions, suggested that binding to, orcross-reactivity of the antibodies of the disclosure with IL-17AF wouldnot necessarily occur. More specifically, the N-terminal region of theIL-17F monomeric subunit of the heterodimer was predicted to stericallyhamper the binding of antibodies of the disclosure to the IL-17AFheterodimer. The expectation was thus that there would not besignificant cross-reactivity for the antibodies with IL-17AF.

However, despite these predictions, we have determined thatcross-binding to IL-17AF by the disclosed antibodies does occur. Thismay in fact be advantageous for a number of reasons. As discussed above,IL-17AF is also implicated as a pro-inflammatory cytokine and may beinvolved in many of the same pathological conditions or undesirablebiological events as described or suspected for IL-17A. The antibodiesof the disclosure may therefore be especially valuable therapeuticallybecause they can target or interfere with both IL-17A and IL-17AF.

Moreover, the present inventors have demonstrated that this bindingbetween the antibodies of the disclosure and IL-17AF also correlates toan inhibition of the biological activity of IL-17AF as observed in invitro assays. Accordingly, the antibodies of the disclosure not onlyefficiently target and antagonize/neutralize the activity of IL-17A, butalso IL-17AF activity as well.

A further unexpected consequence of the work conducted by the presentinventors is as follows. The affinity maturation of the original‘parent’ antibody XAB1 has also resulted in a set of antibodiesretaining a high affinity, or an improved affinity for IL-17A variantsderived from other species such as cynomolgus, rhesus macaque, marmoset,rat, or mouse.

This is unexpected because in striving to improve the affinity of theantibodies of the disclosure for human IL-17A it would not be expectedto also improve the affinity of the resulting antibodies for speciesvariants of IL-17A. In fact, the opposite might normally be expected.Efforts to improve antibody affinity for a specific species variant(i.e. human) of a target antigen would usually be expected to reduceaffinity for other species variants of that antigen. The concept of thespecies variant (or homolog/paralog) recognizes a common ancestry for agiven species, but accepts that divergence has taken place over thecourse of evolutionary history. Accordingly, even where there is a gooddegree of sequence conservation between variants of a particularmolecule that have been identified in different species, it cannot beassumed that an improved affinity for one species variant will have animprovement on the affinity for another species variant. In fact, thedivergence between the sequences for different species generally leadsto the expectation that improvement in affinity for one variant will bemore likely to lead to a reduction (or even abolishment) of the bindingaffinity for another species variant. The sequence identity betweenmouse and human IL-17A is only 62% (Moseley et al. 2003, Cytokine &Growth Factor Reviews 14:155-174).

However, in the present case this was not observed and the antibodyvariants generated by the inventors retained high affinity for IL-17Avariants from other species. This is useful because during the worknecessary to develop a candidate antibody molecule as a usefultherapeutic molecule a variety of tests and assays may be required to becarried out in other species or on cells, molecules or systemscomprising components of, or derived from other species (such ascynomolgus, rhesus macaque, marmoset, rat, or mouse). This makes theantibodies of the disclosure are especially suitable for furtherdevelopment.

Accordingly, antibodies and proteins comprising an antigen-bindingportion thereof as disclosed herein may share a range of desirableproperties including, high affinity for IL-17A, cross-reactivity withIL-17A from other species such as mouse, rat, cynomolgus and marmoset,lack of cross-reactivity for other IL-17 isotypes such as IL-17F, lackof cross-reactivity for other cytokines (such as human or mousecytokines), cross-reactivity with heterodimeric IL-17AF, the ability toblock binding of IL-17A to its receptor such as IL-17RA, the ability toinhibit or neutralize IL-17A induced biological effects such as thestimulation of IL-6 or GRO-alpha secretion, and/or the ability toinhibit in vivo effects mediated by IL-17A (and/or IL-17AF) such as theswelling that is observed in antigen induced arthritis models.

The antibodies and proteins comprising an antigen-binding portionthereof as disclosed herein have also been shown to provide a slowelimination of the antibody-IL17A complex, a slow turnover of the ligandand a long duration of IL17A capture. Further advantageous features ofthese antibodies and proteins are provided in the detailed embodiments.

Homologous Antibodies

In yet another embodiment, an antibody or protein comprising anantigen-binding portion thereof as disclosed herein has full lengthheavy and light chain amino acid sequences; full length heavy and lightchain nucleotide sequences, variable region heavy and light chainnucleotide sequences, or variable region heavy and light chain aminoacid sequences, or all 6 CDR regions amino acid sequences or nucleotidecoding sequences that are homologous to the amino acid or nucleotidesequences of the antibodies XAB1, XAB2, XAB3, XAB4 and XAB5 describedabove, in particular in Table 1, and wherein the antibodies or proteinsof the disclosure retain the desired functional properties of theoriginal XAB1, XAB2, XAB3, XAB4 and XAB5 antibodies.

Desired functional properties of the original XAB1, XAB2, XAB3, XAB4 andXAB5 antibodies may be selected from the group consisting of:

-   -   (i) binding affinity to IL-17A (specific binding to IL-17A), for        example, a K_(D) being 1 nM or less, 100 pM or less, or 10 pM or        less, as measured in the Biacore™ assay, e.g. as described the        Examples;    -   (ii) competitive inhibition of IL-17R binding to IL-17A, for        example, an IC₅₀ being 10 nM or less, or 1 nM or less, or 100 pM        or less, as measured in an in vitro competitive binding assay,        e.g. as described in the Examples;    -   (iii) inhibition of IL-17A dependent activity, for example        production of IL-6 or GRO-alpha, for example, an IC₅₀ being 10        nM or less, or 1 nM or less, or 100 pM or less, as measured in a        cellular assay as described in the Examples;    -   (iv) inhibition of the effects observed, for example knee        swelling, as measured in an in vivo antigen induced arthritis        assay as described in the Examples;    -   (v) cross-reactivity with cynomolgus, rhesus macaque, rat, or        mouse IL-17A polypeptide;    -   (vi) cross-reactivity with human or mouse IL-17AF polypeptide;    -   (vii) binding affinity to IL-17AF (specific binding to IL-17AF),        for example, a K_(D) being 1 nM or less, 100 pM or less, or 10        pM or less, as measured in the Biacore™ assay, e.g. as described        in the Examples;    -   (viii) inhibition of IL-17AF, for example, an IC₅₀ being 200 nM        or less, 150 nM or less, or 100 nM or less, as measured in an in        vitro competitive binding assay as described in the Examples;    -   (ix) suitable properties for drug development, in particular, it        is stable and does not aggregate at in a formulation at high        concentration, i.e., above 50 mg/ml.

For example, the disclosure relates to homologous antibodies of XAB1,XAB2, XAB3, XAB4 and XAB5 (or a protein comprising an antigen-bindingportion thereof), comprising a variable heavy chain (V_(H)) and avariable light chain (V_(L)) sequences where the CDR sequences, i.e. the6 CDR regions; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 or HCDR1′,HCDR2′, HCDR3′, LCDR1′, LCDR2′, LCDR3′, share at least 60, 70, 90, 95 or100 percent sequence identity to the corresponding CDR sequences of atleast one antibody of XAB1, XAB2, XAB3, XAB4 and XAB5, wherein saidhomologous antibody or antigen-binding fragment thereof, such as aprotein comprising an antigen-binding portion thereof, specificallybinds to IL-17A, and the antibody or protein exhibits at least one ofthe following functional properties: it inhibits binding of IL-17A toits receptors, it inhibits IL-17A dependent IL-6 or GRO-alpha productionin cellular assays, or inhibition of the effects observed in an in vivoantigen induced arthritis assay. In a related specific embodiment, thehomologous antibody or protein binds to IL-17A with a K_(D) of 1 nM orless and inhibits binding of IL-17A to its receptors as measured in anin vitro competitive binding assay with an IC₅₀ of 1 nM or less. TheCDRs of XAB1, XAB2, XAB3, XAB4 and XAB5 are defined in the above Table 5and Table 6.

The disclosure further relates to homologous antibodies of XAB1, XAB2,XAB3, XAB4 and XAB5 (or antigen-binding fragments thereof, such as aprotein comprising an antigen-binding portion thereof) comprising aheavy chain variable region and a light chain variable region that areat least 80%, 90%, or at least 95% or 100% identical to thecorresponding heavy and light chain variable regions of any one of theantibodies XAB1, XAB2, XAB3, XAB4 or XAB5, the homologous antibody orprotein specifically binds to IL-17A, and it exhibits at least one ofthe following functional properties: it inhibits binding of IL-17A toits receptor(s), it inhibits IL-17A dependent IL-6 or GRO-alphaproduction in cellular assays, or inhibition of the effects observed inan in vivo antigen induced arthritis assay. In a related specificembodiment, the homologous antibody or antigen binding fragment thereof,such as a protein comprising an antigen-binding portion thereof binds toIL-17A with a K_(D) of 1 nM or less and inhibits binding of IL-17A toits receptor(s), as measured in an in vitro competitive binding assaywith an IC₅₀ of 1 nM or less. The V_(H) and V_(L) amino acid sequencesof XAB1, XAB2, XAB3, XAB4 and XAB5 are defined in the above Table 2.

In another example, the disclosure relates to homologous antibodies ofXAB1, XAB2, XAB3, XAB4 and XAB5 (or antigen-binding fragments thereof,such as a protein comprising an antigen-binding portion thereof)comprising a full length heavy chain and a full length light chain,wherein: the variable heavy chain is encoded by a nucleotide sequencethat is at least 80%, at least 90%, at least 95%, or 100% identical tothe corresponding coding nucleotide sequence of the variable heavy andlight chains of XAB1, XAB2, XAB3, XAB4 and XAB5, the homologous antibodyor antigen-binding fragments thereof, such as a protein comprising anantigen-binding portion thereof, specifically binds to IL-17A, and itexhibits at least one of the following functional properties: itinhibits binding of IL-17A to its receptor(s), it inhibits IL-17Adependent production of IL-6 or GRO-alpha in cellular assays, orinhibition of the effects observed in an in vivo antigen inducedarthritis assay. In a related specific embodiment, the homologousantibody or antigen-binding fragments thereof, such as a proteincomprising an antigen-binding portion thereof binds to IL-17A with aK_(D) of 1 nM or less and inhibits binding to IL-17A as measured in anin vitro competitive binding assay with an IC₅₀ of 1 nM or less. Thecoding nucleotide sequences of the variable regions of XAB1, XAB2, XAB3,XAB4 and XAB5 can be derived from the Table 3 showing the full lengthcoding nucleotide sequences of XAB1, XAB2, XAB3, XAB4 and XAB5 and Table2 showing the amino acid sequences of the variable regions of XAB1,XAB2, XAB3, XAB4 and XAB5.

In various embodiments, the antibody or antigen-binding fragmentthereof, such as a protein comprising an antigen-binding portion of anantibody, may exhibit one or more, two or more, three or more, or fouror more of the desired functional properties discussed above. Theantibody or protein of the disclosure can be, for example, a humanantibody, a humanized antibody or a chimeric antibody. In oneembodiment, the antibody or protein is a fully human silent antibody,such as a fully human silent IgG1 antibody.

Silenced effector functions can be obtained by mutation in the Fcconstant part of the antibodies and have been described in the Art:Strohl 2009 (LALA & N297A); Baudino 2008, D265A (Baudino et al. 2008, J.Immunol. 181:6664-69, Strohl, Colo. 2009, Biotechnology 20:685-91).Examples of silent IgG1 antibodies comprise the so-called LALA mutantcomprising L234A and L235A mutation in the IgG1 Fc amino acid sequence.Another example of a silent IgG1 antibody comprises the D265A mutation.The D265A mutation can also preferably be combined with the P329Amutation (DAPA). Another silent IgG1 antibody comprises the N297Amutation, which results in aglycosylated or non-glycosylated antibodies.

Antibodies with mutant amino acid sequences can be obtained bymutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of thecoding nucleic acid molecules, followed by testing of the encodedaltered antibody for retained function (i.e., the functions set forthabove) using the functional assays described herein.

Antibodies with Conservative Modifications

In certain embodiments, an antibody (or a protein comprisingantigen-binding portion thereof) of the disclosure has a heavy chainvariable region comprising HCDR1, HCDR2, and HCDR3 sequences (or HCDR1′,HCDR2′ and HCDR3′) and a light chain variable region comprising LCDR1,LCDR2, and LCDR3 sequences (or LCDR1′, LCDR2′, and LCDR3′), wherein oneor more of these CDR sequences have specified amino acid sequences basedon the antibodies XAB1, XAB2, XAB3, XAB4 or XAB5 described herein orconservative modifications thereof, and wherein the antibody or proteinretains the desired functional properties of the anti-IL-17A antibodiesof the disclosure.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid substitutions in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one ormore amino acid residues within the CDR regions of an antibody of thedisclosure can be replaced with other amino acid residues from the sameside chain family, and the altered antibody can be tested for retainedfunction using the functional assays described herein.

Modifications can be introduced into an antibody as disclosed herein bystandard techniques known in the art, such as site-directed mutagenesisand PCR-mediated mutagenesis.

Engineered and Modified Antibodies

Other antibodies and antigen-binding fragments, such as proteinscomprising an antigen-binding portion thereof can be prepared using anantibody having one or more of the V_(H) and/or V_(L) sequences of XAB1,XAB2, XAB3, XAB4 or XAB5 shown above as starting material to engineer amodified antibody, which modified antibody may have altered propertiesfrom the starting antibody. An antibody can be engineered by modifyingone or more residues within one or both variable regions (i.e., V_(H)and/or V_(L)), for example within one or more CDR regions and/or withinone or more framework regions. Additionally or alternatively, anantibody can be engineered by modifying residues within the constantregion(s), for example to alter the effector function(s) of theantibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chainscomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. 1998, Nature332:323-327; Jones, P. et al. 1986, Nature 321:522-525; Queen, C. et al.1989, Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.)

Accordingly, another embodiment of the disclosure pertains to anisolated recombinant CDR-grafted anti-IL-17A antibody, comprising the 6CDR regions of any one of XAB1, XAB2, XAB3, XAB4 or XAB5 as defined inTable 5 or Table 6, yet containing different framework sequences fromthe original antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chainsvariable region genes can be found in the “VBase” human germlinesequence database (available on the Internet atwww.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat, E. A., et al. 1991,Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242;Tomlinson, I. M., et al. 1992, J. Mol. Biol. 227:776-798; and Cox, J. P.L. et al. 1994, Eur. J Immunol. 24:827-836.

Examples of framework sequences are those that are structurally similarto the framework sequences used in any one of XAB1, XAB2, XAB3, XAB4 orXAB5. The V_(H) CDR1, 2 and 3 sequences, and the V_(L) CDR1, 2 and 3sequences, can be grafted onto framework regions that have the identicalsequence as that found in the germline immunoglobulin gene from whichthe framework sequence derive, or the CDR sequences can be grafted ontoframework regions that contain one or more mutations as compared to thegermline sequences. For example, it has been found that in certaininstances it is beneficial to mutate residues within the frameworkregions to maintain or enhance the antigen binding ability of theantibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and6,180,370 to Queen et al).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(L) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest, known as “affinity maturation.” Site-directedmutagenesis or PCR-mediated mutagenesis can be performed to introducethe mutation(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assays asdescribed herein and provided in the Examples. Therefore, in oneembodiment, the disclosure relates to affinity matured antibodiesderived from one of XAB1, XAB2, XAB3, XAB4 or XAB5 antibodies.Conservative modifications (as discussed above) can be introduced. Themutations may be amino acid substitutions, additions or deletions.Moreover, typically no more than one, two, three, four or five residueswithin a CDR region are altered. For example, an antibody of thedisclosure is an affinity-matured antibody comprising the 6 CDRs of oneof XAB1, XAB2, XAB3, XAB4 or XAB5 and wherein no more than one, two,three, four or five residues within a CDR region are altered.

Accordingly, in another embodiment, the disclosure provides isolatedengineered anti-IL-17A antibodies comprising a heavy chain variableregion and a light chain variable region which are identical to thecorresponding heavy and light chain variable regions of at least one ofXAB1, XAB2, XAB3, XAB4 or XAB5 antibodies except that the heavy and/orlight chain amino acid sequences of said engineered antibodies containone, two, three, four or five amino acid substitutions, deletions oradditions as compared to the original sequences.

Grafting Antigen-Binding Domains into Alternative Frameworks orScaffolds

A wide variety of antibody/immunoglobulin frameworks or scaffolds can beemployed so long as the resulting polypeptide includes at least onebinding region of XAB1, XAB2, XAB3, XAB4 or XAB5, which specificallybinds to IL-17A. Such frameworks or scaffolds include the 5 mainidiotypes of human immunoglobulins, or fragments thereof (such as thosedisclosed elsewhere herein), and include immunoglobulins of other animalspecies, preferably having humanized aspects. Single heavy-chainantibodies such as those identified in camelids are of particularinterest in this regard. Novel frameworks, scaffolds and fragmentscontinue to be discovered and developed by those skilled in the art.

In one aspect, the disclosure pertains to generating non-immunoglobulinbased antibodies or proteins comprising an antigen-binding portionthereof using non-immunoglobulin scaffolds onto which CDRs of thedisclosure can be grafted. Known or future non-immunoglobulin frameworksand scaffolds may be employed, as long as they comprise a binding regionspecific for the target protein of SEQ ID NO: 76. Such compounds arereferred herein as “polypeptides comprising a target-specific bindingregion”. Examples of non-immunoglobulin framework are further describedin the sections below (camelid antibodies and non-antibody scaffold).

Camelid Antibodies

Antibody proteins obtained from members of the camel and dromedary(Camelus bactrianus and Calelus dromaderius) family including new worldmembers such as llama species (Lama paccos, Lama glama and Lama vicugna)have been characterized with respect to size, structural complexity andantigenicity for human subjects. Certain IgG antibodies from this familyof mammals as found in nature lack light chains, and are thusstructurally distinct from the typical four chain quaternary structurehaving two heavy and two light chains, for antibodies from otheranimals. See PCT Publication No. WO 94/04678.

A region of the camelid antibody which is the small single variabledomain identified as V_(HH) can be obtained by genetic engineering toyield a small protein having high affinity for a target, resulting in alow molecular weight antibody-derived protein known as a “camelidnanobody”. See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see alsoStijlemans, B. et al. 2004, J Biol Chem 279: 1256-1261; Dumoulin, M. etal. 2003, Nature 424: 783-788; Pleschberger, M. et al. 2003,Bioconjugate Chem 14: 440-448; Cortez-Retamozo, V. et al. 2002, Int JCancer 89: 456-62; and Lauwereys, M. et al. 1998, EMBO J 17: 3512-3520.Engineered libraries of camelid antibodies and antibody fragments arecommercially available, for example, from Ablynx, Ghent, Belgium. Aswith other antibodies of non-human origin, an amino acid sequence of acamelid antibody can be altered recombinantly to obtain a sequence thatmore closely resembles a human sequence, i.e., the nanobody can be“humanized”. Thus the natural low antigenicity of camelid antibodies tohumans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth thatof a human IgG molecule and the protein has a physical diameter of onlya few nanometers. One consequence of the small size is the ability ofcamelid nanobodies to bind to antigenic sites that are functionallyinvisible to larger antibody proteins, i.e., camelid nanobodies areuseful as reagents detecting antigens that are otherwise cryptic usingclassical immunological techniques, and as possible therapeutic agents.Thus yet another consequence of small size is that a camelid nanobodycan inhibit as a result of binding to a specific site in a groove ornarrow cleft of a target protein, and hence can serve in a capacity thatmore closely resembles the function of a classical low molecular weightdrug than that of a classical antibody.

The low molecular weight and compact size further result in camelidnanobodies being extremely thermostable, stable to extreme pH and toproteolytic digestion, and poorly antigenic. Another consequence is thatcamelid nanobodies readily move from the circulatory system intotissues, and even cross the blood-brain barrier and can treat disordersthat affect nervous tissue. Nanobodies can further facilitated drugtransport across the blood brain barrier. See U.S. Patent PublicationNo. 20040161738 published Aug. 19, 2004. These features combined withthe low antigenicity to humans indicate great therapeutic potential.Further, these molecules can be fully expressed in prokaryotic cellssuch as E. coli and are expressed as fusion proteins in bacteriophageand are functional.

Engineered nanobodies can further be customized by genetic engineeringto have a half-life in a recipient subject of from 45 minutes to twoweeks. In a specific embodiment, the camelid antibody or nanobody isobtained by grafting the CDRs sequences of the heavy or light chain ofone of the human antibodies of the disclosure, XAB1, XAB2, XAB3, XAB4 orXAB5, into nanobody or single domain antibody framework sequences, asdescribed for example in PCT Publication No. WO 94/04678.

Non-Antibody Scaffold

Known non-immunoglobulin frameworks or scaffolds include, but are notlimited to, Adnectins (fibronectin) (Compound Therapeutics, Inc.,Waltham, Mass.), ankyrin (Molecular Partners AG, Zurich, Switzerland),domain antibodies (Domantis, Ltd (Cambridge, Mass.) and Ablynx nv(Zwijnaarde, Belgium)), lipocalin (Anticalin) (Pieris Proteolab AG,Freising, Germany), small modular immuno-pharmaceuticals (TrubionPharmaceuticals Inc., Seattle, Wash.), maxybodies (Avidia, Inc.(Mountain View, Calif.)), Protein A (Affibody AG, Sweden) and affilin(gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany),protein epitope mimetics (Polyphor Ltd, Allschwil, Switzerland).

(a) Fibronectin Scaffold

The fibronectin scaffolds are based preferably on fibronectin type IIIdomain (e.g., the tenth module of the fibronectin type III (10 Fn3domain)). The fibronectin type III domain has 7 or 8 beta strands whichare distributed between two beta sheets, which themselves pack againsteach other to form the core of the protein, and further containing loops(analogous to CDRs) which connect the beta strands to each other and aresolvent exposed. There are at least three such loops at each edge of thebeta sheet sandwich, where the edge is the boundary of the proteinperpendicular to the direction of the beta strands (U.S. Pat. No.6,818,418).

These fibronectin-based scaffolds are not an immunoglobulin, althoughthe overall fold is closely related to that of the smallest functionalantibody fragment, the variable region of the heavy chain, whichcomprises the entire antigen recognition unit in camel and llama IgG.Because of this structure, the non-immunoglobulin antibody mimicsantigen binding properties that are similar in nature and affinity tothose of antibodies. These scaffolds can be used in a loop randomizationand shuffling strategy in vitro that is similar to the process ofaffinity maturation of antibodies in vivo. These fibronectin-basedmolecules can be used as scaffolds where the loop regions of themolecule can be replaced with CDRs of one of XAB1, XAB2, XAB3, XAB4 orXAB5 using standard cloning techniques.

(b) Ankyrin—Molecular Partners

The technology is based on using proteins with ankyrin derived repeatmodules as scaffolds for bearing variable regions which can be used forbinding to different targets. The ankyrin repeat module is a 33 aminoacid polypeptide consisting of two anti-parallel α-helices and a β-turn.Binding of the variable regions is mostly optimized by using ribosomedisplay.

(c) Maxybodies/Avimers—Avidia

Avimers are derived from natural A-domain containing protein such asLRP-1. These domains are used by nature for protein-protein interactionsand in human over 250 proteins are structurally based on A-domains.Avimers consist of a number of different “A-domain” monomers (2-10)linked via amino acid linkers. Avimers can be created that can bind tothe target antigen using the methodology described in, for example, USPatent Publication Nos 20040175756; 20050053973; 20050048512; and20060008844.

(d) Protein A—Affibody

Affibody® affinity ligands are small, simple proteins composed of athree-helix bundle based on the scaffold of one of the IgG-bindingdomains of Protein A. Protein A is a surface protein from the bacteriumStaphylococcus aureus. This scaffold domain consists of 58 amino acids,13 of which are randomized to generate Affibody® libraries with a largenumber of ligand variants (See e.g., U.S. Pat. No. 5,831,012). Affibody®molecules mimic antibodies; they have a molecular weight of 6 kDa,compared to the molecular weight of antibodies, which is 150 kDa. Inspite of its small size, the binding site of Affibody® molecules issimilar to that of an antibody.

(e) Anticalins—Pieris

Anticalins® are products developed by the company Pieris ProteoLab AG.They are derived from lipocalins, a widespread group of small and robustproteins that are usually involved in the physiological transport orstorage of chemically sensitive or insoluble compounds. Several naturallipocalins occur in human tissues or body liquids.

The protein architecture is reminiscent of immunoglobulins, withhypervariable loops on top of a rigid framework. However, in contrastwith antibodies or their recombinant fragments, lipocalins are composedof a single polypeptide chain with 160 to 180 amino acid residues, beingjust marginally bigger than a single immunoglobulin domain.

The set of four loops, which makes up the binding pocket, showspronounced structural plasticity and tolerates a variety of side chains.The binding site can thus be reshaped in a proprietary process in orderto recognize prescribed target molecules of different shape with highaffinity and specificity.

One protein of lipocalin family, the bilin-binding protein (BBP) ofPieris Brassicae has been used to develop anticalins by mutagenizing theset of four loops. One example of a patent application describing“anticalins” is PCT Publication WO 199916873.

(f) Affilin—Scil Proteins

Affilin™ molecules are small non-immunoglobulin proteins which aredesigned for specific affinities towards proteins and small molecules.New Affilin™ molecules can be very quickly selected from two libraries,each of which is based on a different human derived scaffold protein.

Affilin™ molecules do not show any structural homology to immunoglobulinproteins. Scil Proteins employs two Affilin™ scaffolds, one of which isgamma crystalline, a human structural eye lens protein and the other is“ubiquitin” superfamily proteins. Both human scaffolds are very small,show high temperature stability and are almost resistant to pH changesand denaturing agents. This high stability is mainly due to the expandedbeta sheet structure of the proteins. Examples of gamma crystallinederived proteins are described in WO200104144 and examples of“ubiquitin-like” proteins are described in WO2004106368.

(g) Protein Epitope Mimetics (PEM)

PEM are medium-sized, cyclic, peptide-like molecules (MW 1-2 kDa)mimicking beta-hairpin secondary structures of proteins, the majorsecondary structure involved in protein-protein interactions.

Framework or Fc Engineering

Engineered antibodies and proteins comprising an antigen-binding portionthereof of the disclosure include those in which modifications have beenmade to framework residues within V_(H) and/or V_(L), e.g. to improvethe properties of the antibody. Typically such framework modificationsare made to decrease the immunogenicity of the antibody. For example,one approach is to “backmutate” one or more framework residues to thecorresponding germline sequence. More specifically, an antibody that hasundergone somatic mutation may contain framework residues that differfrom the germline sequence from which the antibody is derived. Suchresidues can be identified by comparing the antibody framework sequencesto the germline sequences from which the antibody is derived. To returnthe framework region sequences to their germline configuration, thesomatic mutations can be “backmutated” to the germline sequence by, forexample, site-directed mutagenesis or PCR-mediated mutagenesis. Such“backmutated” antibodies are also intended to be encompassed by thedisclosure.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell-epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the disclosure may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the disclosure may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below.

As used herein, the term “Fc region” is used to define the C-terminalregion of an immunoglobulin heavy chain, including native sequence Fcregion and variant Fc regions. The human IgG heavy chain Fc region isgenerally defined as comprising the amino acid residue from positionC226 or from P230 to the carboxyl-terminus of the IgG antibody.

The numbering of residues in the Fc region is that of the EU index ofKabat. The C-terminal lysine (residue K447) of the Fc region may beremoved, for example, during production or purification of the antibody.Accordingly, a composition of antibodies of the disclosure may compriseantibody populations with all K447 residues removed, antibodypopulations with no K447 residues removed, and antibody populationshaving a mixture of antibodies with and without the K447 residue.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody or protein comprising anantigen-binding portion thereof is modified to increase its biologicalhalf-life. Various approaches are possible. For example, one or more ofthe following mutations can be introduced: T252L, T254S, T256F, asdescribed in U.S. Pat. No. 6,277,375 to Ward. Alternatively, to increasethe biological half-life, the antibody can be altered within the CH1 orCL region to contain a salvage receptor binding epitope taken from twoloops of a CH2 domain of an Fc region of an IgG, as described in U.S.Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody or protein comprising anantigen-binding portion thereof. For example, one or more amino acidscan be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acidresidues can be replaced with a different amino acid residue such thatthe antibody has altered C1 q binding and/or reduced or abolishedcomplement dependent cytotoxicity (CDC). This approach is described infurther detail in U.S. Pat. No. 6,194,551 by Idusogie et al.

In another embodiment, one or more amino acid residues are altered tothereby alter the ability of the antibody to fix complement. Thisapproach is described further in PCT Publication WO 94/29351 by Bodmeret al.

In yet another embodiment, the Fc region is modified to increase theability of the antibody or protein comprising an antigen-binding portionthereof to mediate antibody dependent cellular cytotoxicity (ADCC)and/or to increase the affinity of the antibody for an Fcγ receptor bymodifying one or more amino acids. This approach is described further inPCT Publication WO 00/42072 by Presta. Moreover, the binding sites onhuman IgG1 for FcγRI, FcγRII, FcγRIII and FcRn have been mapped andvariants with improved binding have been described (see Shields, R. L.et al. 2001, J. Biol. Chem 276:6591-6604).

In certain embodiments, the Fc domain of the IgG1 isotype is used. Insome specific embodiments, a mutant variant of the IgG1 Fc fragment isused, e.g. a silent IgG1 Fc which reduces or eliminates the ability ofthe fusion polypeptide to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to bind to an Fcγ receptor. An example of anIgG1 isotype silent mutant is IgG1 wherein Leucine is replaced byAlanine at amino acid positions 234 and 235 as described by Hezareh etal. 2001, J. Virol 75:12161-8 Another example of an IgG1 isotype silentmutant is IgG1 with D265A mutation (aspartate being substituted byalanine at position 265). In certain embodiments, the Fc domain is asilent Fc mutant preventing glycosylation at position 297 of the Fcdomain. For example, the Fc domain contains an amino acid substitutionof asparagine at position 297. An example of such amino acidsubstitution is the replacement of N297 by a glycine or an alanine.

In still another embodiment, the glycosylation of an antibody or proteincomprising an antigen-binding portion thereof is modified. For example,an aglycoslated antibody can be made (i.e., the antibody lacksglycosylation). Glycosylation can be altered to, for example, increasethe affinity of the antibody for the antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region framework glycosylation sites to therebyeliminate glycosylation at that site. Such aglycosylation may increasethe affinity of the antibody for antigen. Such an approach is describedin further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co et al.

Additionally or alternatively, an antibody or protein comprising anantigen-binding portion thereof can be made that has an altered type ofglycosylation, such as a hypofucosylated antibody having reduced amountsof fucosyl residues or an antibody having increased bisecting GlcNacstructures. Such altered glycosylation patterns have been demonstratedto increase the ADCC ability of antibodies. Such carbohydratemodifications can be accomplished by, for example, expressing theantibody in a host cell with altered glycosylation machinery. Cells withaltered glycosylation machinery have been described in the art and canbe used as host cells in which to express recombinant antibodies of thedisclosure to thereby produce an antibody with altered glycosylation.For example, EP 1 176 195 by Hang et al. describes a cell line with afunctionally disrupted FUT8 gene, which encodes a fucosyl transferase,such that antibodies expressed in such a cell line exhibithypofucosylation. Therefore, in one embodiment, the antibodies of thedisclosure are produced by recombinant expression in a cell line whichexhibits a hypofucosylation pattern, for example, a mammalian cell linewith deficient expression of the FUT8 gene encoding fucosyltransferase.PCT Publication WO 03/035835 by Presta describes a variant CHO cellline, Lecl3 cells, with reduced ability to attach fucose toAsn(297)-linked carbohydrates, also resulting in hypofucosylation ofantibodies expressed in that host cell (see also Shields, R. L. et al.2002, J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 byUmana et al. describes cell lines engineered to expressglycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-Nacetylglucosaminyltransferase III (GnTIII)) such that antibodiesexpressed in the engineered cell lines exhibit increased bisectingGlcNac structures which results in increased ADCC activity of theantibodies (see also Umana et al. 1999, Nat. Biotech. 17:176-180).Alternatively, the antibodies and proteins comprising an antigen-bindingportion thereof of the disclosure can be produced in a yeast or afilamentous fungus engineered for mammalian-like glycosylation pattern,and capable of producing antibodies lacking fucose as glycosylationpattern (see for example EP 1 297 172).

Another modification of the antibodies and proteins comprising anantigen-binding portion thereof as disclosed herein that is contemplatedby the disclosure is pegylation. These molecules can be pegylated to,for example, increase their biological (e.g., serum) half-life. Forexample, to pegylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.The pegylation can be carried out by an acylation reaction or analkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-010) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the disclosure. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

Another modification of the antibodies and proteins comprising anantigen-binding portion thereof that is contemplated by the disclosureis a conjugate or a protein fusion of at least the antigen-bindingregion of the antibody of the disclosure to serum protein, such as humanserum albumin or a fragment thereof to increase half-life of theresulting molecule. Such approach is for example described in Ballanceet al. EP 0 322 094.

Another possibility is a fusion of at least the antigen-binding regionof the antibody of the disclosure to proteins capable of binding toserum proteins, such human serum albumin to increase half-life of theresulting molecule. Such approach is for example described in Nygren etal., EP 0 486 525.

Methods of Engineering Altered Antibodies

As discussed above, the anti-IL-17A antibodies having V_(H) and V_(L)sequences or full length heavy and light chain sequences shown hereincan be used to create new anti-IL-17A antibodies by modifying fulllength heavy chain and/or light chain sequences, V_(H) and/or V_(L)sequences, or the constant region(s) attached thereto. Thus, in anotheraspect of the disclosure, the structural features of an anti-IL-17Aantibody of the disclosure are used to create structurally relatedanti-IL-17A antibodies or protein comprising an antigen-binding portionthereof that retain at least one functional property of the antibodiesdisclosed herein, such as binding to human IL-17A and also inhibitingone or more functional properties of IL-17A (e.g., inhibiting binding ofIL-17A or IL-17AF to its receptor(s), inhibiting IL-17A or IL-17AFinduced production of IL-6, GRO-alpha, etc.) inhibitory activity in invivo assays.

Other antibodies retaining substantially the same binding properties toIL-17A include chimeric antibodies or CDR grafted antibodies of any oneof XAB1, XAB2, XAB3, XAB4 or XAB5 which retain the same VH and VLregions, or the CDR regions, of any one of XAB1, XAB2, XAB3, XAB4 orXAB5 and different constant regions or framework regions (for example adifferent Fc region selected from a different isotype, for example IgG4or IgG2).

For example, one or more CDR regions of any one of XAB1, XAB2, XAB3,XAB4 or XAB5, or mutations thereof, can be combined recombinantly withknown framework regions and/or other CDRs to create additional,recombinantly-engineered, anti-IL-17A antibodies of the disclosure, asdiscussed above. Other types of modifications include those described inthe previous section. The starting material for the engineering methodis one or more of the V_(H) and/or V_(L) sequences of XAB1, XAB2, XAB3,XAB4 or XAB5 provided in the tables above, or one or more CDR regionthereof. To create the engineered antibody, it is not necessary toactually prepare (i.e., express as a protein) an antibody having one ormore of the V_(H) and/or V_(L) sequences of XAB1, XAB2, XAB3, XAB4 orXAB5, or one or more CDR regions thereof. Rather, the informationcontained in the sequence(s) is used as the starting material to createa “second generation” sequence(s) derived from the original sequence(s)and then the “second generation” sequence(s) is prepared and expressedas a protein.

The second generation sequences are derived for example by altering theDNA coding sequence of at least one amino acid residue within the heavychain variable region antibody sequence and/or the light chain variableregion antibody sequence of any one of XAB1, XAB2, XAB3, XAB4 or XAB5,to create at least one altered antibody sequence; and expressing thealtered antibody sequence as a protein.

Accordingly, in another embodiment, the disclosure provides a method forpreparing an anti-IL-17A antibody optimized for expression in amammalian cell consisting of: a full length heavy chain antibodysequence a full length light chain antibody sequence of any one of XAB1,XAB2, XAB3, XAB4 or XAB5; altering at least one codon in the nucleotidecoding sequence, said codon encoding an amino acid residue within thefull length heavy chain antibody sequence and/or the full length lightchain antibody sequence to create at least one altered antibodysequence; and expressing the altered antibody sequence as a protein.

The altered antibody sequence can also be prepared by screening antibodylibraries having unique heavy and light CDR3 sequences of any one ofXAB1, XAB2, XAB3, XAB4 or XAB5 respectively, or minimal essentialbinding determinants as described in US Patent Publication No.20050255552, and alternative sequences for CDR1 and CDR2 sequences. Thescreening can be performed according to any screening technologyappropriate for screening antibodies from antibody libraries, such asphage display technology.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence. The antibody encoded by the alteredantibody sequence(s) is one that retains one, some or all of the desiredfunctional properties of the anti-IL-17A antibodies described herein,which functional properties include, but are not limited to,specifically binding to human IL-17A; and/or it inhibits binding ofIL-17A to its receptor(s); and/or it inhibits IL-17A induced productionof, for example, IL-6 or GRO-alpha etc.

The altered antibody may exhibit one or more, two or more, or three ormore of the functional properties discussed above.

In certain embodiments of the methods of engineering antibodies of thedisclosure, mutations can be introduced randomly or selectively alongall or part of an anti-IL-17A antibody coding sequence and the resultingmodified anti-IL-17A antibodies can be screened for binding activityand/or other functional properties as described herein. Mutationalmethods have been described in the art. For example, PCT Publication WO02/092780 by Short describes methods for creating and screening antibodymutations using saturation mutagenesis, synthetic ligation assembly, ora combination thereof. Alternatively, PCT Publication WO 03/074679 byLazar et al. describes methods of using computational screening methodsto optimize physiochemical properties of antibodies.

Nucleic Acid Molecules Encoding Antibodies of the Disclosure

Another aspect of the disclosure pertains to nucleic acid molecules thatencode the antibodies or proteins of the disclosure. Examples ofvariable light chain nucleotide sequences are those encoding thevariable light chain amino acid sequences of any one of XAB1, XAB2,XAB3, XAB4 and XAB5, the latter sequences being derived from the Table 3(showing the entire nucleotide coding sequences of heavy and lightchains of XAB1, XAB2, XAB3, XAB4 or XAB5) and Table 2 (showing the aminoacid sequences of the variable regions of XAB1, XAB2, XAB3, XAB4 orXAB5).

The disclosure also pertains to nucleic acid molecules that derive fromthe latter sequences having been optimized for protein expression inmammalian cells, for example, CHO cell lines.

The nucleic acids may be present in whole cells, in a cell lysate, ormay be nucleic acids in a partially purified or substantially pure form.A nucleic acid is “isolated” or “rendered substantially pure” whenpurified away from other cellular components or other contaminants,e.g., other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, CsCl banding, column chromatography,agarose gel electrophoresis and others well known in the art. See, F.Ausubel, et al., ed. 1987, Current Protocols in Molecular Biology,Greene Publishing and Wiley Interscience, New York. A nucleic acid ofthe disclosure can be, for example, DNA or RNA and may or may notcontain intronic sequences. In an embodiment, the nucleic acid is a cDNAmolecule. The nucleic acid may be present in a vector such as a phagedisplay vector, or in a recombinant plasmid vector.

Nucleic acids of the disclosure can be obtained using standard molecularbiology techniques. Once DNA fragments encoding, for example, VH andV_(L) segments are obtained, these DNA fragments can be furthermanipulated by standard recombinant DNA techniques, for example toconvert the variable region genes to full-length antibody chain genes,to Fab fragment genes or to an scFv gene. In these manipulations, aV_(L)- or V_(H)-encoding DNA fragment is operatively linked to anotherDNA molecule, or to a fragment encoding another protein, such as anantibody constant region or a flexible linker. The term “operativelylinked”, as used in this context, is intended to mean that the two DNAfragments are joined in a functional manner, for example, such that theamino acid sequences encoded by the two DNA fragments remain in-frame,or such that the protein is expressed under control of a desiredpromoter.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g., Kabat, E. A., el al. 1991, Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region. In some embodiments,the heavy chain constant region is selected among IgG1 isotypes. For aFab fragment heavy chain gene, the V_(H)-encoding DNA can be operativelylinked to another DNA molecule encoding only the heavy chain CH1constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as to a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabat,E. A., et al. 1991, Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. The light chain constantregion can be a kappa or a lambda constant region.

To create an scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly4-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and VH regions joined by the flexible linker (see e.g.,Bird et al. 1988, Science 242:423-426; Huston et al. 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883; McCafferty et al. 1990, Nature348:552-554).

Isolation of Recombinant Antibodies of the Disclosure

A variety of methods of screening antibodies and proteins comprising anantigen-binding portion thereof have been described in the Art. Suchmethods may be divided into in vivo systems, such as transgenic micecapable of producing fully human antibodies upon antigen immunizationand in vitro systems, consisting of generating antibody DNA codinglibraries, expressing the DNA library in an appropriate system forantibody production, selecting the clone that express antibody candidatethat binds to the target with the affinity selection criteria andrecovering the corresponding coding sequence of the selected clone.These in vitro technologies are known as display technologies, andinclude without limitation, phage display, RNA or DNA display, ribosomedisplay, yeast or mammalian cell display. They have been well describedin the Art (for a review see for example: Nelson et al. 2010, NatureReviews Drug discovery, “Development trends for human monoclonalantibody therapeutics” (Advance Online Publication) and Hoogenboom etal. 2001, Method in Molecular Biology 178:1-37, O'Brien et al., ed.,Human Press, Totowa, N.J.). In one specific embodiment, humanrecombinant antibodies of the disclosure are isolated using phagedisplay methods for screening libraries of human recombinant antibodylibraries, such as HuCAL® libraries.

Repertoires of V_(H) and V_(L) genes or related CDR regions can beseparately cloned by polymerase chain reaction (PCR) or synthesized byDNA synthesizer and recombined randomly in phage libraries, which canthen be screened for antigen-binding clones. Such phage display methodsfor isolating human antibodies are established in the art or describedin the examples below. See for example: U.S. Pat. Nos. 5,223,409;5,403,484; and U.S. Pat. No. 5,571,698 to Ladner et al.; U.S. Pat. Nos.5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404;6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

In a certain embodiment, human antibodies directed against IL-17A can beidentified using transgenic or transchromosomic mice carrying parts ofthe human immune system rather than the mouse system. These transgenicand transchromosomic mice include mice referred to herein as HuMAb miceand KM mice, respectively, and are collectively referred to herein as“human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode un-rearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.1994, Nature 368:856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al. 1994, supra; reviewed in Lonberg, N.,1994 Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. 1995, Intern. Rev. Immunol. 13:65-93, and Harding, F. andLonberg, N. 1995, Ann. N. Y. Acad. Sci. 764:536-546). The preparationand use of HuMAb mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al. 1992, Nucleic AcidsResearch 20:6287-6295; Chen, J. et al. 1993, International Immunology5:647-656; Tuaillon et al. 1993, Proc. Natl. Acad. Sci. USA94:3720-3724; Choi et al. 1993, Nature Genetics 4:117-123; Chen, J. etal. 1993, EMBO J. 12: 821-830; Tuaillon et al. 1994, J. Immunol.152:2912-2920; Taylor, L. et al. 1994, International Immunology 579-591;and Fishwild, D. et al. 1996, Nature Biotechnology 14: 845-851. Seefurther, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429;all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCTPublication Nos. WO 92103918, WO 93/12227, WO 94/25585, WO 97113852, WO98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT PublicationNo. WO 01/14424 to Korman et al.

In another embodiment, human antibodies of the disclosure can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-IL-17A antibodies of the disclosure. For example, an alternativetransgenic system referred to as the Xenomouse from Abgenix, Inc. can beused. Such mice are described in, e.g., U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6, 150,584 and 6,162,963 to Kucherlapati et al. Aswill be appreciated by a person skilled in the art, several other mousemodels may be used, such as the Trianni™ mouse from Trianni, Inc., theVeloclmmune™ mouse from Regeneron Pharmaceuticals, Inc., or the Kymouse™mouse from Kymab Limited.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-IL-17A antibodies of the disclosure. For example, mice carryingboth a human heavy chain transchromosome and a human light chaintranschromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al. 2000, Proc. Natl. Acad. Sci. USA97:722-727.

Human monoclonal antibodies of the disclosure can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Generation of Monoclonal Antibodies of the Disclosure from the MurineSystem

Monoclonal antibodies (mAbs) can be produced by a variety of techniques,including conventional monoclonal antibody methodology e.g., thestandard somatic cell hybridization technique of Kohler and Milstein1975, Nature 256:495. Many techniques for producing monoclonal antibodycan be employed e.g., viral or oncogenic transformation of Blymphocytes.

An animal system for preparing hybridomas is the murine system.Hybridoma production in the mouse is a well-established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present disclosure can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art. See e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al.

Generation of Hybridomas Producing Human Monoclonal Antibodies

To generate hybridomas producing human monoclonal antibodies of thedisclosure, splenocytes and/or lymph node cells from immunized mice canbe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific or epitope-specific antibodies. Forexample, single cell suspensions of splenic lymphocytes from immunizedmice can be fused to one-sixth the number of P3X63-Ag8.653 nonsecretingmouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cells are plated atapproximately 2×145 in flat bottom microtiter plates, followed by a twoweek incubation in selective medium containing 20% fetal Clone Serum,18% “653” conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mMsodium pyruvate, 5 mM HEPES, 0:055 mM 2-mercaptoethanol, 50 units/mlpenicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and 1×HAT (Sigma;the HAT is added 24 hours after the fusion). After approximately twoweeks, cells can be cultured in medium in which the HAT is replaced withHT. Individual wells can then be screened by ELISA for human monoclonalIgM and IgG antibodies. Once extensive hybridoma growth occurs, mediumcan be observed usually after 10-14 days. The antibody secretinghybridomas can be replated, screened again, and if still positive forhuman IgG, the monoclonal antibodies can be subcloned once or twice bylimiting dilution. The stable subclones can then be cultured in vitro togenerate small amounts of antibody in tissue culture medium forcharacterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

Generation of Transfectomas Producing Monoclonal Antibodies

Antibodies of the disclosure can be produced in a host cell transfectomausing, for example, a combination of recombinant DNA techniques and genetransfection methods as is well known in the art (e.g., Morrison, S.1985, Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains can beobtained by standard molecular biology or biochemistry techniques (e.g.,DNA chemical synthesis, PCR amplification or cDNA cloning using ahybridoma that expresses the antibody of interest) and the DNAs can beinserted into expression vectors such that the genes are operativelylinked to transcriptional and translational control sequences. In thiscontext, the term “operatively linked” is intended to mean that anantibody gene is ligated into a vector such that transcriptional andtranslational control sequences within the vector serve their intendedfunction of regulating the transcription and translation of the antibodygene. The expression vector and expression control sequences are chosento be compatible with the expression host cell used. The antibody lightchain gene and the antibody heavy chain gene can be inserted intoseparate vector or, more typically, both genes are inserted into thesame expression vector. The antibody genes are inserted into theexpression vector by standard methods (e.g., ligation of complementaryrestriction sites on the antibody gene fragment and vector, or blunt endligation if no restriction sites are present). The light and heavy chainvariable regions of the antibodies described herein can be used tocreate full-length antibody genes of any antibody isotype by insertingthem into expression vectors already encoding heavy chain constant andlight chain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the C_(H) segment(s) within the vectorand the V_(L) segment is operatively linked to the C_(L) segment withinthe vector. Additionally or alternatively, the recombinant expressionvector can encode a signal peptide, also called leader sequence, whichfacilitates secretion of the antibody chain from a host cell. Theantibody chain gene can be cloned into the vector such that the signalpeptide is linked in frame to the amino terminus of the antibody chaingene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein). Examples of such signal peptides are foundin Table 7, and examples of polynucleotide sequences coding for thesignal peptides are found in Table 8.

TABLE 7 Signal peptides for heavy and light peptide chains. SignalSequence Used for heavy or peptide ID no. light peptide chain 1 SEQ IDNO: 59 Heavy 2 SEQ ID NO: 60 Light 3 SEQ ID NO: 63 Heavy 4 SEQ ID NO: 64Light 5 SEQ ID NO: 67 Heavy 6 SEQ ID NO: 68 Light

TABLE 8 Polynucleotide sequences coding for the signal peptides. Codingfor Coding signal peptide signal peptide sequence for heavy or light no.Sequence ID no. chain 1 SEQ ID NO: 61 Heavy 2 SEQ ID NO: 62 Light 3 SEQID NO: 65 Heavy 4 SEQ ID NO: 66 Light 5 SEQ ID NO: 69 Heavy 6 SEQ ID NO:70 Light

In addition to the antibody chain genes, the recombinant expressionvectors of the disclosure carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel 1990,Gene Expression Technology. Methods in Enzymology 185, Academic Press,San Diego, Calif.). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Regulatory sequences for mammalian host cell expression includeviral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., theadenovirus major late promoter (AdMLP)), and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or P-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SRa promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al. 1988, Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the disclosure may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, standard techniques wereapplied to transfect a host cell with the expression vector(s) encodingthe heavy and light chains. The various forms of the term “transfection”are intended to encompass a wide variety of techniques commonly used forthe introduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. It is theoretically possible toexpress the antibodies of the disclosure in either prokaryotic oreukaryotic host cells. Expression of antibodies in eukaryotic cells, forexample mammalian host cells, yeast or filamentous fungi, is discussedbecause such eukaryotic cells, and in particular mammalian cells, aremore likely than prokaryotic cells to assemble and secrete a properlyfolded and immunologically active antibody.

In one specific embodiment, a cloning or expression vector according tothe disclosure comprises either at least one of coding sequences fromTable 3, operatively linked to suitable promoter sequences. In anotherspecific embodiment, a cloning or expression vector according to thedisclosure comprises either at least one of coding sequences from Table4, operatively linked to suitable promoter sequences.

Mammalian host cells for expressing the recombinant antibodies of thedisclosure include Chinese Hamster Ovary (CHO cells) (including dhfr-CHOcells, described Urlaub and Chasin 1980, Proc. Natl. Acad. Sci. USA77:4216-4220 used with a DH FR selectable marker, e.g., as described inR. J. Kaufman and P. A. Sharp 1982, Mol. Biol. 159:601-621), CHOK1 dhfr+cell lines, NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another expression system is the GS geneexpression system shown in PCT Publications WO 87/04462, WO 89/01036 andEP 0 338 841. In one embodiment, mammalian host cells for expressing therecombinant antibodies of the disclosure include mammalian cell linesdeficient for FUT8 gene expression, for example as described in U.S.Pat. No. 6,946,292.

When recombinant expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods (See for example Abhinav et al. 2007,Journal of Chromatography 848:28-37).

In one specific embodiment, the host cell of the disclosure is a hostcell transfected with an expression vector having the coding sequencesselected from the group consisting of SEQ ID NO: 18, 31, 51, 19, 28, 32,38, 40, 46, 48, 52, 56, and 58, suitable for the expression of XAB1,XAB2, XAB3, XAB4 or XAB5 respectively, operatively linked to suitablepromoter sequences.

The latter host cells may then be further cultured under suitableconditions for the expression and production of an antibody of thedisclosure selected from the group consisting of XAB1, XAB2, XAB3, XAB4or XAB5 respectively.

Immunoconjugates

In another aspect, the present disclosure features an anti-IL-17Aantibody of the disclosure, or a fragment thereof, conjugated to anactive or therapeutic moiety, such as a cytotoxin, a drug (e.g., animmunosuppressant) or a radiotoxin. Such conjugates are referred toherein as “immunoconjugates”. This may be particularly preferred ifIL-17A is expressed on the surface of Th17 cells (Brucklacher-Waldert etal. 2009, J Immunol. 183:5494-501).

Immunoconjugates that include one or more cytotoxins are referred to as“immunotoxins.” A cytotoxin or cytotoxic agent includes any agent thatis detrimental to (e.g., kills) cells. Examples include taxon,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, t. colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents also include, for example,antimetabolites (e.g., methotrexate, β-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), ablating agents (e.g.,mechlorethamine, thioepa chloraxnbucil, meiphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Other examples of therapeutic cytotoxins that can be conjugated to anantibody of the disclosure include duocarmycins, calicheamicins,maytansines and auristatins, and derivatives thereof. An example of acalicheamicin antibody conjugate is commercially available (Mylotarg™;Wyeth-Ayerst).

Cytoxins can be conjugated to antibodies of the disclosure using linkertechnology available in the art. Examples of linker types that have beenused to conjugate a cytotoxin to an antibody include, but are notlimited to, hydrazones, thioethers, esters, disulfides andpeptide-containing linkers. A linker can be chosen that is, for example,susceptible to cleavage by low pH within the lysosomal compartment orsusceptible to cleavage by proteases, such as proteases preferentiallyexpressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito, G. et al.2003, Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et al. 2003, CancerImmunol. Immunother. 52:328-337; Payne, G. 2003, Cancer Cell 3:207-212;Allen, T. M. 2002, Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman,R. J. 2002, Curr. Opin. Investig. Drugs 3:1089-1091; Senter, P. D. andSpringer, C. J. 2001, Adv. Drug Deliv. Rev. 53:247-264.

Antibodies of the present disclosure also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹,yttrium⁹⁰, and lutetium¹⁷⁷. Method for preparing radioimmunconjugatesare established in the art. Examples of radioimmunoconjugates arecommercially available, including Zevalin™ (DEC Pharmaceuticals) andBexxar™ (Corixa Pharmaceuticals), and similar methods can be used toprepare radioimmunoconjugates using the antibodies of the disclosure.

The antibody conjugates of the disclosure can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL1”), interleukin-2(“IL2”), interleukin-6 (“IL6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al. 1985, “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56; Hellstrom et al.1987, “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), pp. 623-53 Thorpe 1985, “Antibody CarriersOf Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies '84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506; Thorpe et al. 1982, Immunol. Rev., 62:119-58.

Bispecific Molecules

In another aspect, the present disclosure features bispecific ormultispecific molecules comprising an anti-IL-17A/AF antibody or proteincomprising an antigen-binding portion thereof of the disclosure. Anantibody or protein of the disclosure can be derivatized or linked toanother functional molecule, e.g., another peptide or protein (e.g.,another antibody or ligand for a receptor) to generate a bispecificmolecule that binds to at least two different binding sites or targetmolecules. The antibody or protein of the disclosure may in fact bederivatized or linked to more than one other functional molecule togenerate multi-specific molecules that bind to more than two differentbinding sites and/or target molecules; such multi-specific molecules arealso intended to be encompassed by the term “bispecific molecule” asused herein. To create a bispecific molecule of the disclosure, anantibody or protein of the disclosure can be functionally linked (e.g.,by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other binding molecules, such as anotherantibody, antibody fragment, peptide or binding mimetic, such that abispecific molecule results.

Accordingly, the present disclosure includes bispecific moleculescomprising at least one first binding specificity for IL-17A, forexample, one antigen-binding portion of any one of XAB1, XAB2, XAB3,XAB4 or XAB5 and a second binding specificity for a second targetepitope. For example, the second target epitope is another epitope ofIL-17A different from the first target epitope. Another example is abispecific molecule comprising at least one first binding specificityfor IL-17A, for example, one antigen-binding portion of any one of XAB1,XAB2, XAB3, XAB4 or XAB5 and a second binding specificity for an epitopeelsewhere within IL-17A or within another target antigen.

Additionally, for the disclosure in which the bispecific molecule ismulti-specific, the molecule can further include a third bindingspecificity, in addition to the first and second target epitope.

In one embodiment, the bispecific molecules of the disclosure compriseas a binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778.

Other antibodies which can be employed in the bispecific molecules ofthe disclosure are murine, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present disclosure can be prepared byconjugating the constituent binding specificities, using methods knownin the art. For example, each binding-specificity of the bispecificmolecule can be generated separately and then conjugated to one another.When the binding specificities are proteins or peptides, a variety ofcoupling or cross-linking agents can be used for covalent conjugation.

Examples of cross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. 1984, J. Exp. Med. 160:1686;Liu, M A et al. 1985, Proc. Natl. Acad. Sci. USA 82:8648). Other methodsinclude those described in Paulus 1985, Behring Ins. Mitt. No. 78,118-132; Brennan et al. 1985, Science 229:81-83), and Glennie et al.1987, J. Immunol. 139: 2367-2375). Conjugating agents are SATA andsulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugated bysulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particular embodiment, the hinge region is modified tocontain an odd number of sulfhydryl residues, for example one, prior toconjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand x Fab fusion protein. A bispecific molecule of thedisclosure can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. Nos.5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;5,013,653; 5,258,498; and 5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (REA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest.

Multivalent Antibodies

In another aspect, the present disclosure provides multivalentantibodies comprising at least two identical or differentantigen-binding portions of the antibodies of the disclosure binding toIL-17A, for example, selected from antigen-binding portions of any oneof XAB1, XAB2, XAB3, XAB4 or XAB5. In one embodiment, the multivalentantibodies provide at least two, three or four antigen-binding portionsof the antibodies. The antigen-binding portions can be linked togethervia protein fusion or covalent or non-covalent linkage. Alternatively,methods of linkage have been described for the bispecific molecules.Tetravalent compounds can be obtained for example by cross-linkingantibodies of the disclosure with an antibody that binds to the constantregions of the antibodies of the disclosure, for example the Fc or hingeregion.

Pharmaceutical Compositions

In another aspect, the present disclosure provides a composition, e.g.,a pharmaceutical composition, containing one or a combination ofantibodies or proteins comprising an antigen-binding portion thereof ofthe present disclosure, for example, one antibody selected from thegroup consisting of XAB1, XAB2, XAB3, XAB4 and XAB5, formulated togetherwith a pharmaceutically acceptable carrier. Such compositions mayinclude one or a combination of (e.g., two or more different)antibodies, or immunoconjugates or bispecific molecules of thedisclosure. For example, a pharmaceutical composition of the disclosurecan comprise a combination of antibodies or proteins that bind todifferent epitopes on the target antigen or that have complementaryactivities.

Pharmaceutical compositions of the disclosure also can be administeredin combination therapy, i.e., combined with other agents. For example,the combination therapy can include an anti-IL-17A antibody or proteinof the present disclosure, for example one antibody selected from thegroup consisting of XAB1, XAB2, XAB3, XAB4 and XAB5, combined with atleast one other anti-inflammatory or another chemotherapeutic agent, forexample, an immunosuppressant agent. Examples of therapeutic agents thatcan be used in combination therapy are described in greater detail belowin the section on uses of the antibodies or proteins of the disclosure.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. The carrier should be suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). In oneembodiment, the carrier should be suitable for subcutaneous route.Depending on the route of administration, the active compound, i.e.,antibody, immunoconjugate, or bispecific molecule, may be coated in amaterial to protect the compound from the action of acids and othernatural conditions that may inactivate the compound.

The pharmaceutical compositions of the disclosure may include one ormore pharmaceutically acceptable salts. A “pharmaceutically acceptablesalt” refers to a salt that retains the desired biological activity ofthe parent compound and does not impart any undesired toxicologicaleffects (see e.g., Berge, S. M., et al. 1977, J. Pharm. Sci. 66:1-19).Examples of such salts include acid addition salts and base additionsalts. Acid addition salts include those derived from nontoxic inorganicacids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,hydroiodic, phosphorous and the like, as well as from nontoxic organicacids such as aliphatic mono- and di-carboxylic acids,phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromaticacids, aliphatic and aromatic sulfonic acids and the like. Base additionsalts include those derived from alkaline earth metals, such as sodium,potassium, magnesium, calcium and the like, as well as from nontoxicorganic amines, such as N,N′-dibenzylethylenediamine, N-methylglucamine,chloroprocaine, choline, diethanolamine, ethylenediamine, procaine andthe like.

A pharmaceutical composition of the disclosure also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include:

water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures and by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas, aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe disclosure is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In many cases, one can include isotonic agents, forexample, sugars, polyalcohols such as mannitol, sorbitol, or sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent that delays absorption for example, monostearate salts andgelatin.

Reviews on the development of stable protein (e.g. antibody)formulations may be found in Cleland et al. 1993, Crit. Reviews. Ther.Drug Carrier Systems 10(4):307-377 and Wei Wang 1999, Int. J.Pharmaceutcs 185:129-88. Additional formulation discussions forantibodies may be found, e.g., in Daugherty and Mrsny 2006, AdvancedDrug Delivery Reviews 58: 686-706; U.S. Pat. Nos. 6,171,586, 4,618,486,US Publication No. 20060286103, PCT Publication WO 06/044908, WO07/095337, WO 04/016286, Colandene et al. 2007, J. Pharm. Sci 96:1598-1608; Schulman 2001, Am. J. Respir. Crit. Care Med. 164:S6-S11 andother known references.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include one or more of the following components: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents, antibacterial agents such as benzyl alcohol ormethyl parabens, antioxidants such as ascorbic acid or sodium bisulfite,chelating agents such ethylenediaminetetraacetic acid, buffers such asacetates, citrates or phosphates, and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Suchpreparations may be enclosed in ampoules, disposables syringes ormultiple dose vials made of glass or plastic.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the antibodies or proteins of the disclosure into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, the methodsof preparation are vacuum drying and freeze-drying (lyophilization) thatyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

In one specific embodiment, the antibodies XAB1, XAB2, XAB3, XAB4 orXAB5 were administered as a liquid formulation in a vial. The amount ofdrug per vial was 150 mg. The liquid contained 150 mg/mL antibody, 4.8mM L-Histidine, 15.2 mM L-Histidine-HCl 220 mM Sucrose and 0.04%Polysorbate 20, at pH 6.0±0.5. A 20% overfill was added to permitcomplete removal of the intended dose.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, from about 0.1percent to about 70 percent, or from about 1 percent to about 30 percentof active ingredient in combination with a pharmaceutically acceptablecarrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of sensitivity in individuals.

For administration of the antibody or protein, the dosage ranges fromabout 0.0001 to 150 mg/kg, such as 5, 15, and 50 mg/kg subcutaneousadministration, and more usually 0.01 to 5 mg/kg, of the host bodyweight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg bodyweight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weightor within the range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once per month, once every 3 months oronce every three to 6 months. Dosage regimens for an anti-IL-17Aantibody or protein of the disclosure include 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg, 10 mg/kg, 20 mg/kg or 30 mg/kg byintravenous administration, with the antibody being given using one ofthe following dosing schedules: every four weeks for six dosages, thenevery three months; every three weeks; 3 mg/kg body weight once followedby 1 mg/kg body weight every three weeks.

In some methods, two or more antibodies with different bindingspecificities are administered simultaneously, in which case the dosageof each antibody administered falls within the ranges indicated.Antibodies or proteins of the disclosure are usually administered onmultiple occasions. Intervals between single dosages can be, forexample, weekly, monthly, every three months or yearly. Intervals canalso be irregular as indicated by measuring blood levels of antibody tothe target antigen in the patient. In some methods, dosage is adjustedto achieve a plasma antibody concentration of about 1-1000 μg/ml and insome methods about 25-300 μg/ml.

Alternatively, antibody or protein can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of theantibody in the patient. In general, human antibodies show the longesthalf-life, followed by humanized antibodies, chimeric antibodies, andnonhuman antibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated or until the patient shows partial or completeamelioration of symptoms of disease. Thereafter, the patient can beadministered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present disclosure may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentdisclosure employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of an anti-IL-17A antibody orprotein of the disclosure can result in a decrease in severity ofdisease symptoms, an increase in frequency and duration of diseasesymptom-free periods, or a prevention of impairment or disability due tothe disease affliction.

A composition of the present disclosure can be administered by one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Routes of administration for antibodies of the disclosureinclude intravenous, intramuscular, intradermal, intraperitoneal,subcutaneous, spinal or other parenteral routes of administration, forexample by injection or infusion. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrastemal injection andinfusion.

Alternatively, an antibody or protein of the disclosure can beadministered by a non-parenteral route, such as a topical, epidermal ormucosal route of administration, for example, intranasally, orally,vaginally, rectally, sublingually or topically.

The antibodies or proteins of the disclosure can be prepared withcarriers that will protect the antibodies against rapid release, such asa controlled release formulation, including implants, transdermalpatches, and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Many methods for the preparation of such formulationsare patented or generally known to those skilled in the art. See, e.g.,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson,ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in one embodiment, a therapeutic composition ofthe disclosure can be administered with a needleless hypodermicinjection device, such as the devices shown in U.S. Pat. Nos. 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.Examples of well-known implants and modules useful in the presentdisclosure include: U.S. Pat. No. 4,487,603, which shows an implantablemicro-infusion pump for dispensing medication at a controlled rate; U.S.Pat. No. 4,486,194, which shows a therapeutic device for administeringmedicants through the skin; U.S. Pat. No. 4,447,233, which shows amedication infusion pump for delivering medication at a precise infusionrate; U.S. Pat. No. 4,447,224, which shows a variable flow implantableinfusion apparatus for continuous drug delivery; U.S. Pat. No.4,439,196, which shows an osmotic drug delivery system havingmulti-chamber compartments; and U.S. Pat. No. 4,475,196, which shows anosmotic drug delivery system. Many other such implants, deliverysystems, and modules are known to those skilled in the art.

In certain embodiments, the antibodies or proteins of the disclosure canbe formulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds of the disclosure cross the BBB(if desired), they can be formulated, for example, in liposomes. Formethods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;5,374,548; and 5,399,331. The liposomes may comprise one or moremoieties which are selectively transported into specific cells ororgans, thus enhance targeted drug delivery (see, e.g., V. V. Ranade1989, J. Cline Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al. 1988, Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. 1995, FEBS Lett. 357:140; M.Owais et al. 1995, Antimicrob. Agents Chernother. 39:180); surfactantprotein A receptor (Briscoe et al. 1995, Am. J. Physiol.1233:134); p120(Schreier et al. 1994, J. Biol. Chem. 269:9090); see also Keinanen andLaukkanen 1994, FEBS Lett. 346:123; Killion andFidler 1994,Immunomethods 4:273.

Uses and Methods of the Disclosure

The antibodies or proteins of the present disclosure have in vitro andin vivo diagnostic and therapeutic utilities. For example, thesemolecules can be administered to cells in culture, e.g. in vitro or invivo, or in a subject, e.g., in vivo, to treat, prevent or diagnose avariety of disorders.

The methods are particularly suitable for treating, preventing ordiagnosing IL-17A-related disorders and/or autoimmune and inflammatorydisorders, e.g., rheumatoid arthritis, or psoriasis.

Specifically, the disclosure provides a method of treatingIL-17A-related disorders and/or autoimmune and inflammatory disorders.In certain embodiments the method comprises the step of administeringisolated antibody or protein comprising an antigen-binding portionthereof, according to the disclosure, to a subject in need thereof.

The disclosure also provides methods for decreasing or suppressingIL-17A or IL-17AF induced signaling response in target cells or tissuesby contacting a cell with a composition comprising a therapeuticallyeffective dose of the antibodies of the disclosure.

The disclosure also provides methods for decreasing levels of IL6,CXCL1, IL-8, GM-CSF and/or CCL2 in a cell comprising the step ofcontacting a cell with antibody or antigen binding fragment, such as aprotein comprising an antigen-binding portion thereof, according to thedisclosure.

In the present description the phrase “IL-17A/AF mediated disease” or“IL-17A/AF-related disorder” encompasses all diseases and medicalconditions in which IL-17A or IL-17AF plays a role, whether directly orindirectly, in the disease or medical condition, including thecausation, development, progress, persistence or pathology of thedisease or condition. Accordingly these terms include conditionsassociated with or characterized by aberrant IL-17A/AF levels and/ordiseases or conditions that can be treated by reducing or suppressingIL-17A/AF induced activity in target cells or tissues e.g. theproduction of IL-6 or GRO-alpha. These include inflammatory conditionsand autoimmune diseases, such as arthritis, rheumatoid arthritis, orpsoriasis. These further include allergies and allergic conditions,hypersensitivity reactions, chronic obstructive pulmonary disease,cystic fibrosis and organ or tissue transplant rejection.

For example, the antibodies or proteins of the disclosure may be usedfor the treatment of recipients of heart, lung, combined heart-lung,liver, kidney, pancreatic, skin or corneal transplants, includingallograft rejection or xenograft rejection, and for the prevention ofgraft-versus-host disease, such as following bone marrow transplant, andorgan transplant associated arteriosclerosis.

The antibodies or proteins of the disclosure, whilst not being limitedto, are useful for the treatment, prevention, or amelioration ofautoimmune disease and of inflammatory conditions, in particularinflammatory conditions with an aetiology including an autoimmunecomponent such as arthritis (for example rheumatoid arthritis, arthritischronica progrediente and arthritis deformans) and rheumatic diseases,including inflammatory conditions and rheumatic diseases involving boneloss, inflammatory pain, spondyloarhropathies including ankylosingspondylitis, Reiter syndrome, reactive arthritis, psoriatic arthritis,juvenile idiopathic arthritis and enterophathis arthritis, enthesitis,hypersensitivity (including both airways hypersensitivity and dermalhypersensitivity) and allergies. Specific auto-immune diseases for whichantibodies of the disclosure may be employed include autoimmunehaematological disorders (including e.g. hemolytic anaemia, aplasticanaemia, pure red cell anaemia and idiopathic thrombocytopenia),systemic lupus erythematosus (SLE), lupus nephritis, inflammatory musclediseases (dermatomyosytis), periodontitis, polychondritis, scleroderma,Wegener granulomatosis, dermatomyositis, chronic active hepatitis,myasthenia gravis, psoriasis, Steven-Johnson syndrome, idiopathic sprue,autoimmune inflammatory bowel disease (including e.g. ulcerativecolitis, Crohn's disease and irritable bowel syndrome), endocrineophthalmopathy, Graves' disease, sarcoidosis, multiple sclerosis,systemic sclerosis, fibrotic diseases, primary biliary cirrhosis,juvenile diabetes (diabetes mellitus type I), uveitis,keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitiallung fibrosis, periprosthetic osteolysis, glomerulonephritis (with andwithout nephrotic syndrome, e.g. including idiopathic nephrotic syndromeor minimal change nephropathy), multiple myeloma other types of tumors,inflammatory disease of skin and cornea, myositis, loosening of boneimplants, metabolic disorders, (such as obesity, atherosclerosis andother cardiovascular diseases including dilated cardiomyopathy,myocarditis, diabetes mellitus type II, and dyslipidemia), andautoimmune thyroid diseases (including Hashimoto thyroiditis), small andmedium vessel primary vasculitis, large vessel vasculitides includinggiant cell arteritis, hidradenitis suppurativa, neuromyelitis optica,Sjögren's syndrome, Behcet's disease, atopic and contact dermatitis,bronchiolitis, inflammatory muscle diseases, autoimmune peripheralneurophaties, immunological renal, hepatic and thyroid diseases,inflammation and atherothrombosis, autoinflammatory fever syndromes,immunohematological disorders, and bullous diseases of the skin andmucous membranes. Anatomically, uveitis can be anterior, intermediate,posterior, or pan-uveitis. It can be chronic or acute. The etiology ofuveitis can be autoimmune or non-infectious, infectious, associated withsystemic disease, or a white-dot syndrome.

The antibodies or proteins of the disclosure may also be useful for thetreatment, prevention, or amelioration of asthma, bronchitis,bronchiolitis, idiopathic interstitial pneumonias, pneumoconiosis,pulmonary emphysema, and other obstructive or inflammatory diseases ofthe airways.

The antibodies or proteins of the disclosure may also be useful fortreating diseases of bone metabolism including osteoarthritis,osteoporosis and other inflammatory arthritis, and bone loss in general,including age-related bone loss, and in particular periodontal disease.

In addition, the antibodies or proteins of the disclosure may also beuseful for treating chronic candidiasis and other chronic fungaldiseases, as well as complications of infections with parasites, andcomplications of smoking are considered to be promising avenues oftreatment, as well as viral infection and complications of viralinfection.

Inhibition of IL-17 and its receptor is among the most promising newmodes of actions (MOA) for the treatment of chronic inflammatorydiseases, with psoriasis being the most advanced indication amongseveral diseases currently being studied for IL-17 modulator drugdevelopment (Miossec P and Kolls J K. 2012, Nat Rev Drug Discov.10:763-76).

Several studies have unambiguously demonstrated that blocking IL-17A inpatients with moderate to severe plaque psoriasis is safe in the shortterm and induces very remarkable improvements (e.g. Hueber W, Patel D D,Dryja T, et al 2010, Sci Transl Med.; 2:52ra72). These findings exceededexpectations and confirmed the hypothesis that IL-17A is a key signalingmolecule in the pathogenesis of psoriasis (Garber K. 2012, NatBiotechnology 30:475-477).

Furthermore, in several animal models including the most common multiplesclerosis (MS) model experimental autoimmune encephalomyelitis, IL-17 ispivotal in the inflammatory processes (Bettelli E, et al 2008, Nature;453:1051-57, Wang H H, et al 2011, J Clin Neurosci; 18(10):1313-7,Matsushita T, et al 2013, PLoS One; 8(4):e64835). IL-17 effects aremainly proinflammatory, and synergize with other cytokines. IL-17effects such as induction of chemokine production by epithelial cells,upregulation of interleukin (IL)-1b, tumor necrosis factor alpha (TNFa)and matrix metalloproteinase (MMP)-9 in macrophages, and induction ofthe secretion of IL 6, IL-8 and prostaglandin E2, fit well with manyaspects of the MS pathology. There is also data arguing against apivotal role of IL-17 in neuroinflammation, including transgenicoverexpression models in mice (Haak S et al 2009, JCI, 119:61-69).

Asthma is a heterogeneous inflammatory disease of the airways that ismanifested clinically by symptoms of airflow obstruction that varies inseverity either spontaneously or in response to treatment. While asthmahas been considered to be driven by T helper cell type 2 (Th2) cells andtheir products, recent data suggest that a Th2-high gene signature ispresent in the airways of only ˜50% of subjects with asthma (Woodruff PG et al 2009, Am J Respir Crit Care Med 180:388-95). Neutrophilicinflammation is dominant in acute severe asthma; some individuals withasthma present with prominent sputum neutrophilia and a poor clinicalresponse to inhaled steroids; and sputum neutrophilia is prominent inasthmatic individuals taking large doses of inhaled and/or oral steroids(Wenzel 2012, Nature Med 18:716-25).

Increased levels of IL-17A that correlate with the severity of asthmahave been reported in the circulation and airways of individuals withasthma compared to healthy controls. Pre-clinical studies in mousemodels of allergic pulmonary inflammation have implicated a requirementfor IL-17A and its receptor (IL-17RA) in neutrophilic airwayinflammation and steroid-resistant airway hyper responsiveness. Thus,the properties of IL-17A in vitro, its presence in increased amounts inasthma, and the pre-clinical models of the disease support a role forIL-17A in neutrophilic and/or Th2-low forms of the disease that arepoorly responsive to steroids (Cosmi L et al 2009, Am J Respir Crit CareMed 180:388-95).

Thus, the following list of conditions comprises particularly preferredtargets for treatment with antibodies or proteins comprising anantigen-binding portion thereof according to the disclosure: Multiplesclerosis, psoriasis, asthma, systemic lupus erythematosus (SLE), andlupus nephritis.

The antibodies or proteins of the disclosure may be administered as thesole active ingredient or in conjunction with, e.g. as an adjuvant to orin combination to, other drugs e.g. immunosuppressive orimmunomodulating agents or other anti-inflammatory agents or othercytotoxic or anti-cancer agents, e.g. for the treatment or prevention ofdiseases mentioned above. For example, the antibodies of the disclosuremay be used in combination with DMARD, e.g. Gold salts, sulphasalazine,antimalarias, methotrexate, D-penicillamine, azathioprine, mycophenolicacid, tacrolimus, sirolimus, minocycline, leflunomide, glucocorticoids;a calcineurin inhibitor, e.g. cyclosporin A or FK 506; a modulator oflymphocyte recirculation, e.g. FTY720 and FTY720 analogs; a mTORinhibitor, e.g. rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CC1779,ABT578, AP23573 or TAFA-93; an ascomycin having immuno-suppressiveproperties, e.g. ABT-281, ASM981, etc.; corticosteroids;cyclophosphamide; azathioprine; leflunomide, mizoribine; myco-pheno-latemofetil; 15-deoxyspergualine or an immunosuppressive homologue, analogueor derivative thereof; immunosuppressive monoclonal antibodies, e.g.,monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4,CD7, CD8, CD25, CD28, CD40. CD45, CD58, CD80, CD86 or their ligands;other immunomodulatory compounds, e.g. a recombinant binding moleculehaving at least a portion of the extracellular domain of CTLA4 or amutant thereof, e.g. an at least extracellular portion of CTLA4 or amutant thereof joined to a non-CTLA4 protein sequence, e.g. CTLA4lg (forex. designated ATCC 68629) or a mutant thereof, e.g. LEA29Y; adhesionmolecule inhibitors, e.g. LFA-1 antagonists, ICAM-1 or -3 antagonists,VCAM-4 antagonists or VLA-4 antagonists; or a chemotherapeutic agent,e.g. paclitaxel, gemcitabine, cisplatinum, doxorubicin or5-fluorouracil; anti TNF agents, e.g. monoclonal antibodies to TNF, e.g.infliximab, adalimumab, CDP870, or receptor constructs to TNF-RI orTNF-RII, e.g. Etanercept, PEG-TNF-RI, blockers of proinflammatorycytokines, IL1 blockers, e.g. Anakinra or IL1 trap, canakinumab, IL13blockers, IL4 blockers, IL6 blockers, other IL17 blockers (such assecukinumab, broadalumab, ixekizumab); chemokines blockers, e.g.,inhibitors or activators of proteases, e.g. metalloproteases, anti-IL15antibodies, anti-IL6 antibodies, anti-IL4 antibodies, anti-IL13antibodies, anti-CD20 antibodies, NSAIDs, such as aspirin or ananti-infectious agent (list not limited to the agent mentioned).

In accordance with the foregoing the present disclosure provides in ayet further aspect:

A method as defined above comprising co-administration, e.g.concomitantly or in sequence, of a therapeutically effective amount ofan anti-IL-17A antibody or protein comprising an antigen-binding portionthereof as disclosed herein, and at least one second drug substance,said second drug substance being a immuno-suppressive/immunomodulatory,anti-inflammatory chemotherapeutic or anti-infectious drug, e.g. asindicated above.

Or, a therapeutic combination, e.g. a kit, comprising of atherapeutically effective amount of a) an antibody or protein or proteincomprising an antigen-binding portion thereof as disclosed herein, andb) at least one second substance selected from animmuno-suppressive/immunomodulatory, anti-inflammatory chemotherapeuticor anti-infectious drug, e.g. as indicated above. The kit may compriseinstructions for its administration.

Where the antibodies or proteins comprising an antigen-binding portionthereof as disclosed herein are administered in conjunction with otherimmuno-suppressive/immunomodulatory, anti-inflammatory chemotherapeuticor anti-infectious therapy, dosages of the co-administered combinationcompound will of course vary depending on the type of co-drug employed,e.g. whether it is a DMARD, anti-TNF, IL1 blocker or others, on thespecific drug employed, on the condition being treated and so forth.

In one embodiment, the antibodies or proteins comprising anantigen-binding portion thereof can be used to detect levels of IL-17A,or levels of cells that contain IL-17A. This can be achieved, forexample, by contacting a sample (such as an in vitro sample) and acontrol sample with the anti-IL-17A antibody (or protein) underconditions that allow for the formation of a complex between theantibody and IL-17A. Any complexes formed between the antibody (orprotein) and IL-17A are detected and compared in the sample and thecontrol. For example, standard detection methods, well known in the art,such as ELISA and flow cytometric assays, can be performed using thecompositions of the disclosure.

Accordingly, in one aspect, the disclosure further provides methods fordetecting the presence of IL-17A (e.g., human IL-17A antigen) in asample, or measuring the amount of IL-17A, comprising contacting thesample, and a control sample, with an antibody or protein of thedisclosure, or an antigen-binding portion thereof, which specificallybinds to IL-17A, under conditions that allow for formation of a complexbetween the antibody or portion thereof and IL-17A. The formation of acomplex is then detected, wherein a difference in complex formationbetween the sample and the control sample is indicative of the presenceof IL-17A in the sample.

Also within the scope of the disclosure are kits consisting of thecompositions (e.g., antibodies, proteins, human antibodies andbispecific molecules) of the disclosure and instructions for use. Thekit can further contain a least one additional reagent, or one or moreadditional antibodies or proteins of the disclosure (e.g., an antibodyhaving a complementary activity which binds to an epitope on the targetantigen distinct from the first antibody). Kits typically include alabel indicating the intended use of the contents of the kit. The termlabel includes any writing, or recorded material supplied on or with thekit, or which otherwise accompanies the kit. The kit may furthercomprise tools for diagnosing whether a patient belongs to a group thatwill respond to an anti-IL-17A antibody treatment, as defined above.

The disclosure having been fully described is now further illustrated bythe following examples and claims, which are illustrative and are notmeant to be further limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a space-filling representation of the XAB1 Fab. FIG. 1B is acartoon representation of the XAB1 Fab.

FIG. 2A shows the two XAB1 Fv fragments in space-filling representationof the XAB1 Fv complex with human IL-17A and FIG. 2B shows the two XAB1Fv fragments in cartoon representation of the XAB1 Fv complex with humanIL-17A.

FIG. 3A is a graph showing the normalized signal versus the FabConcentration (M).

FIG. 3B is a graph showing the normalized remaining signal versus thewashing incubation time (hours). FIG. 3C is a graph showing thenormalized signal versus the Fab competitor concentration (M). Thesegraphs are ELISA results according to an example. The graph numberingcorresponds to the candidate designation as follows: 1 is MB440; 2 isMB464; 3 is MB468; 4 is MB444; 5 is MB435; 6 is MB463; 7 is XAB1.

FIG. 4A shows the two XAB2 Fv fragments in complex with human IL-17A inspace-filling representation and FIG. 4B shows the two XAB2 Fv fragmentsin complex with human IL-17A in cartoon representation.

FIG. 5 provides the three-dimensional structure of the XAB2 Fv complexwith human IL-17A as a close-up view.

FIG. 6A shows the two XAB5 Fv fragments in complex with human IL-17A inspace-filling representation and FIG. 6B shows the two XAB5 Fv fragmentsin complex with human IL-17A in cartoon representation.

FIG. 7 provides the three-dimensional structure of the XAB5 Fv complexwith human IL-17A, as a close-up view of the antibody L-CDR1.

FIG. 8A shows the two XAB4 Fv fragments in complex with human IL-17A inspace-filling representation FIG. 8B shows the two XAB4 Fv fragments incomplex with human IL-17A in cartoon representation.

FIG. 9 provides the three-dimensional structure of the XAB4 Fv complexwith human IL-17A as a close-up view of the antibody L-CDR1.

FIG. 10 provides the three-dimensional structure of the XAB4 Fv complexwith human IL-17A as a close-up view of the antibody L-CDR2.

FIG. 11 is a graph showing the therapeutic score for XAB4 in anexperimental autoimmune encephalomyelitis (EAE) model.

FIG. 12 is a graph showing the therapeutic weight change (%) of animalsin the EAE model.

FIG. 13 is a graph showing the cumulative therapeutic scores in the EAEmodel.

FIG. 14 shows a comparison of the therapeutic score pre- andpost-treatment in the EAE model.

FIG. 15A show the therapeutic score pre- and post-treatment in the EAEmodel, with XAB4. FIG. 15B show the therapeutic score pre- andpost-treatment in the EAE model, with vehicle

FIG. 16 is a graph showing the prophylactic score for XAB4 in the EAEmodel.

FIG. 17 is a graph showing the prophylactic weight change (%) of animalsin the EAE model.

FIG. 18 is a graph showing the cumulative prophylactic scores in the EAEmodel.

FIG. 19 is a graph showing the maximum prophylactic scores in the EAEmodel.

FIG. 20 is a graph showing the EAE onset in the EAE model.

FIG. 21A shows antagonism of TNF-α or IL-17A stimulation, orIL-17A/TNF-α co-stimulation by XAB4, by IL-6. FIG. 21B shows antagonismof IL-1β or IL-17A/IL-1β co-stimulation by XAB4, by IL-6. The model usedis a human astrocyte model.

FIG. 22A shows antagonism of TNF-α or IL-17A stimulation, orIL-17A/TNF-α co-stimulation by XAB4, by CXCL1. FIG. 22B shows antagonismof IL-1β or IL-17A/IL-1β co-stimulation by XAB4, by CXCL1. The modelused is a human astrocyte model.

FIG. 23A shows antagonism of TNF-α or IL-17A stimulation, orIL-17A/TNF-α co-stimulation by XAB4, by IL8. FIG. 23B shows antagonismof IL-1β or IL-17A/IL-1β co-stimulation by XAB4, by IL8. The model usedis in a human astrocyte model.

FIG. 24A shows antagonism of TNF-α or IL-17A stimulation, orIL-17A/TNF-α co-stimulation by XAB4, by GM-CSF. FIG. 24B showsantagonism of IL-1β or IL-17A/IL-1β co-stimulation by XAB4, by GM-CSF.The model used is a human astrocyte model.

FIG. 25A shows antagonism of TNF-α or IL-17A stimulation, orIL-17A/TNF-α co-stimulation by XAB4, by CCL2. FIG. 25B shows antagonismof IL-1β or IL-17A/IL-1β co-stimulation by XAB4, by CCL2. The model usedis a human astrocyte model.

EXAMPLES

XAB1 is a human IgG1/κ monoclonal antibody. It was generated usingstandard molecular biological techniques. In brief, the Medarex systemwas used. Mice were immunized with recombinant human IL-17A. Mice wereeuthanized by CO₂ inhalation and spleen cells were harvested and fusedwith a myeloma cell line using PEG 4000. Fused cells were plated intowells with a feeder layer of peritoneal cells. Supernatants were takenfrom cultured cells and assayed for IL-17A reactive mAbs by ELISA.Clones positive for the production of IL-17A mAbs were selected andplated out.

The hybridoma responsible for the secretion of XAB1 was identified forfurther characterization on the basis of initial promisingantibody/antigen binding characteristics such as binding affinity forIL-17A, ability to block IL-17A binding to its receptor, and ability toblock IL-17A mediated biological effects in in vitro assays.

The amino acid sequence of XAB1 is SEQ ID NO: 14 (heavy chain) and SEQID NO: 15 (light chain). XAB1 was chosen for subsequent affinitymaturation.

As a first step toward structure-guided affinity maturation, the crystalstructure of the XAB1 Fab in the free state as well as the correspondingFv complex with human IL-17A were determined as described below. Theanalysis of the three-dimensional structure of the XAB1 Fv complex withhuman IL-17A allowed for a rational affinity maturation process to becarried out alongside, and as an alternative to, a more randomisedprocess. Further details are provided below.

In addition, X-ray crystallography was used to characterise some of theaffinity matured variant antibodies that were generated. Analysis ofcrystal data from the affinity matured variants allowed for a deeperunderstanding of the binding behaviour of the variant antibodies andsome unexpected properties were discovered as will be described furtherbelow.

Example 1. Crystal Structure of the XAB1 Fab in the Free State

(i) Material and Methods

Standard molecular biological protocols were used to obtain the XAB1 Fabantibody fragment. In brief, the Fab was cloned and expressed in E. coliW3110 with a C-terminal hexahistidine tag on the heavy-chain. Therecombinant protein was purified by Ni-chelate chromatography followedby size-exclusion chromatography on a SPX-75 column in 10 mM TRIS pH7.4, 25 mM NaCl. The XAB1 Fab was then concentrated by ultra-filtrationto 10.4 mg/ml and crystallized.

Standard crystallization protocols were followed. In brief, crystalswere grown at 19° C. in SD2 96 well-plates, using the method of vapourdiffusion in sitting drops. The protein stock was mixed 1:1 with acrystallization buffer containing 40% PEG 300, 0.1M sodiumphosphate-citrate pH 4.2. Total drop size was 0.4 μl. Prior to X-raydata collection, one crystal was mounted in a nylon cryo-loop anddirectly flash cooled into liquid nitrogen.

X-ray data collection and processing was carried out using standardprotocols. Briefly, X-ray data to 2.1 Å resolution were collected at theSwiss Light Source, beamline X10SA, with a MAR225 CCD detector, using1.0000 Å X-ray radiation. In total, 180 images of 1.0° oscillation eachwere recorded at a crystal-to-detector distance of 190 mm and processedwith the HKL2000 software package. The crystal belonged to space groupC2 with cell parameters a=51.63 Å, b=132.09 Å, c=77.25 Å, α=90.00°,β=98.88°, γ=90.00° and one XAB1 Fab molecule in the asymmetric unit.R-sym to 2.1 Å resolution was 10.4% and data completeness 99.0%.

The structure was determined by molecular replacement with the programPHASER. Search models for the V_(H)/V_(L) and C_(H1)/C_(L) domains weregenerated from PDB entry 1 HEZ. Iterative model building and refinementwere performed with the programs Coot (Crystallographic Object-OrientedToolkit) and CNX (Crystallography & NMR eXplorer) version 2002, until nofurther significant improvements could be made to the model. Final R-and R-free for all data were 0.188 and 0.231, respectively. The finalrefined model showed a root-mean-square deviation (RMSD) from ideal bondlengths and bond angles of 0.004 Å and 0.9°, respectively.

(ii) Results

The results of the X-ray refinement of the XAB1 Fab are provided in

Table 9 and the three-dimensional structure is shown in FIG. 1.

TABLE 9 X-ray refinement of the XAB1 Fab with the program CNX. REMARK 3REMARK 3 REFINEMENT. REMARK 3 PROGRAM: CNX 2002 REMARK 3 AUTHORS:Brunger, Adams, Clore, Delano, REMARK 3     Gros, Grosse-Kunstleve,Jiang, REMARK 3     Kuszewski, Nilges, Pannu, Read, REMARK 3     Rice,Simonson, Warren REMARK 3     and REMARK 3     Accelrys Inc., REMARK 3    Yip, Dzakula). REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3RESOLUTION RANGE HIGH (ANGSTROMS): 2.10 REMARK 3 RESOLUTION RANGE LOW(ANGSTROMS): 33.33 REMARK 3 DATA CUTOFF (SIGMA(F)): 0.0 REMARK 3 DATACUTOFF HIGH (ABS(F)): 19645630.62 REMARK 3 DATA CUTOFF LOW (ABS(F)):0.000000 REMARK 3 COMPLETENESS (WORKING + TEST) (%): 98.2 REMARK 3NUMBER OF REFLECTIONS: 29298 REMARK 3 REMARK 3 FIT TO DATA USED INREFINEMENT. REMARK 3 CROSS-VALIDATION METHOD: THROUGHOUT REMARK 3 FREE RVALUE TEST SET SELECTION: RANDOM REMARK 3 R VALUE (WORKING SET): 0.188REMARK 3 FREE R VALUE: 0.231 REMARK 3 FREE R VALUE TEST SET SIZE (%):4.9 REMARK 3 FREE R VALUE TEST SET COUNT: 1436 REMARK 3 ESTIMATED ERROROF FREE R VALUE: 0.006 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTIONBIN. REMARK 3 TOTAL NUMBER OF BINS USED: 6 REMARK 3 BIN RESOLUTION RANGEHIGH (A): 2.10 REMARK 3 BIN RESOLUTION RANGE LOW (A): 2.23 REMARK 3 BINCOMPLETENESS (WORKING + TEST) (%): 94.7 REMARK 3 REFLECTIONS IN BIN(WORKING SET): 4478 REMARK 3 BIN R VALUE (WORKING SET): 0.201 REMARK 3BIN FREE R VALUE: 0.241 REMARK 3 BIN FREE R VALUE TEST SET SIZE (%): 4.5REMARK 3 BIN FREE R VALUE TEST SET COUNT: 213 REMARK 3 ESTIMATED ERROROF BIN FREE R VALUE: 0.016 REMARK 3 REMARK 3 NUMBER OF NON-HYDROGENATOMS USED IN REFINEMENT. REMARK 3 PROTEIN ATOMS: 3311 REMARK 3 NUCLEICACID ATOMS: 0 REMARK 3 HETEROGEN ATOMS: 5 REMARK 3 SOLVENT ATOMS: 313REMARK 3 REMARK 3 B VALUES. REMARK 3 FROM WILSON PLOT (A**2): 21.1REMARK 3 MEAN B VALUE (OVERALL, A**2): 27.4 REMARK 3 OVERALL ANISOTROPICB VALUE. REMARK 3 B11 (A**2): −6.02 REMARK 3 B22 (A**2): 3.30 REMARK 3B33 (A**2): 2.73 REMARK 3 B12 (A**2): 0.00 REMARK 3 B13 (A**2): 3.82REMARK 3 B23 (A**2): 0.00 REMARK 3 REMARK 3 BULK SOLVENT MODELING.REMARK 3 METHOD USED: FLAT MODEL REMARK 3 KSOL: 0.399279 REMARK 3 BSOL:54.4727 (A**2) REMARK 3 REMARK 3 ESTIMATED COORDINATE ERROR. REMARK 3ESD FROM LUZZATI PLOT (A): 0.21 REMARK 3 ESD FROM SIGMAA (A): 0.12REMARK 3 LOW RESOLUTION CUTOFF (A): 5.00 REMARK 3 REMARK 3CROSS-VALIDATED ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM C-VLUZZATI PLOT (A): 0.29 REMARK 3 ESD FROM C-V SIGMAA (A): 0.14 REMARK 3REMARK 3 RMS DEVIATIONS FROM IDEAL VALUES. REMARK 3 BOND LENGTHS (A):0.004 REMARK 3 BOND ANGLES (DEGREES): 0.9 REMARK 3 DIHEDRAL ANGLES(DEGREES): 21.4 REMARK 3 IMPROPER ANGLES (DEGREES): 0.58 REMARK 3 REMARK3 ISOTROPIC THERMAL MODEL: RESTRAINED REMARK 3 REMARK 3 ISOTROPICTHERMAL FACTOR RESTRAINTS. RMS SIGMA REMARK 3 MAIN-CHAIN BOND (A**2):1.41; 1.50 REMARK 3 MAIN-CHAIN ANGLE (A**2): 2.21; 2.00 REMARK 3SIDE-CHAIN BOND (A**2): 2.31; 2.00 REMARK 3 SIDE-CHAIN ANGLE (A**2):3.44; 2.50 REMARK 3 REMARK 3 NCS MODEL: NONE REMARK 3 REMARK 3 NCSRESTRAINTS. RMS SIGMA/WEIGHT REMARK 3 GROUP 1 POSITIONAL (A): NULL; NULLREMARK 3 GROUP 1 B-FACTOR (A**2): NULL; NULL REMARK 3 REMARK 3 PARAMETERFILE 1: protein_rep.param REMARK 3 PARAMETER FILE 2: water_rep.paramREMARK 3 TOPOLOGY FILE 1: protein_no_ctertop REMARK 3 TOPOLOGY FILE 2:water.top REMARK 3 REMARK 3 OTHER REFINEMENT REMARKS: NULL SSBOND 1 CYSL 23 CYS L 88 SSBOND 2 CYS L 134 CYS L 194 SSBOND 3 CYS H 22 CYS H 96SSBOND 4 CYS H 143 CYS H 199 CRYST1 51.627 132.089 77.247 90.00 98.8890.00 C 1 2 1  8 ORIGX1 1.000000 0.000000 0.000000 0.00000 ORIGX20.000000 1.000000 0.000000 0.00000 ORIGX3 0.000000 0.000000 1.0000000.00000 SCALE1 0.019370 0.000000 0.003027 0.00000 SCALE2 0.0000000.007571 0.000000 0.00000 SCALE3 0.000000 0.000000 0.013103 0.00000

FIG. 1 provides the three-dimensional structure of the XAB1 Fab asobtained in Example 1. FIG. 1A is a space-filling representation. FIG.1B is a cartoon representation. The heavy- and lightchain of the XAB1Fab appears in dark and light grey, respectively.

Example 2. Crystal Structure of the XAB1 Fv Complex with Human IL-17A:Analysis of the Paratope for Structure-Guided Affinity Maturation

-   -   (i) Material and Methods

Standard molecular biological protocols were used to obtain the XAB1 Fvantibody fragment. In brief, the Fv was cloned and expressed in E. coliW3110 with a C-terminal hexahistidine tag on the heavy-chain and aC-terminal Strep-tag on the light-chain. The recombinant protein waspurified by Ni-chelate chromatography.

The XAB1 Fv fragment complex with human IL-17A was then prepared usingstandard methodology. In brief, human IL-17A (1.1 mg) was mixed with anexcess of Fv (2.7 mg) and the complex was run on a S100 size-exclusionchromatography, in 10 mM TRIS pH 7.4, 25 mM NaCl. The protein complexwas then concentrated by ultra-filtration to 21.2 mg/ml andcrystallized.

Standard crystallization protocols were followed. In brief, crystalswere grown at 19° C. in SD2 96 well-plates, using the method of vapourdiffusion in sitting drops. The protein stock was mixed 1:1 with acrystallization buffer containing 10% PEG 20,000, 0.1M Bicine pH 9.0,2.0% (v/v) dioxane. Total drop size was 0.4 μl. Prior to X-ray datacollection, one crystal was briefly transferred into a 1:1 mix of thecrystallization buffer with 20% PEG 20,000, 30% glycerol, and then flashcooled into liquid nitrogen.

X-ray data collection and processing was carried out using standardprotocols. Briefly, X-ray data to 3.0 Å resolution were collected at theSwiss Light Source, Beamline X10SA, with a MAR225 CCD detector, using1.0000 Å X-ray radiation. In total, 110 images of 1.0° oscillation eachwere recorded at a crystal-to-detector distance of 300 mm and processedwith the HKL2000 software package. The crystal belonged to space groupP2₁2₁2 with cell parameters a=184.31 Å, b=55.81 Å, c=70.99 Å, α=β=γ=90°.R-sym to 3.0 Å resolution was 11.2% and data completeness 99.9%.

The structure was determined by molecular replacement with the programPHASER. A search model for the XAB1 Fv was generated from the crystalstructure of the XAB1 Fab previously determined (see Example 1). Asearch model for IL-17A was generated from the published human IL-17Fcrystal structure (PDB entry 1jpy). Iterative model building andrefinement were performed with Coot (Crystallographic Object-OrientedToolkit) and CNX (Crystallography & NMR eXplorer) version 2002, until nofurther significant improvements could be made to the model. Final R-and R-free for all data were 0.215 and 0.269, respectively. The finalrefined model showed a root-mean-square deviation (RMSD) from ideal bondlengths and bond angles of 0.007 Å and 1.0°, respectively.

(ii) Results

The molecular replacement calculations revealed a dimeric complexcomprising one IL-17A homodimer with two XAB1 Fv fragments bound. Theresults of the X-ray refinement of the XAB1 Fv complex with human IL-17Aare provided in Table 10 and the three-dimensional structure of thiscomplex is shown in FIG. 2. Each XAB1 Fv makes contacts to both IL-17Asubunits, but the vast majority of the intermolecular contacts (about96% of the buried surface) are contributed by one IL-17A subunit only.

TABLE 10 X-ray refinement of the XAB1 Fv complex with IL-17A obtained bythe program CNX. REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM: CNX2002 REMARK 3 AUTHORS: Brunger, Adams, Clore, Delano, REMARK 3     Gros,Grosse-Kunstleve, Jiang, REMARK 3     Kuszewski, Nilges, Pannu, Read,REMARK 3     Rice, Simonson, Warren REMARK 3     and REMARK 3    Accelrys Inc., REMARK 3     (Badger, Berard, Kumar, Szalma, REMARK 3    Yip, Dzakula). REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3RESOLUTION RANGE HIGH (ANGSTROMS): 3.01 REMARK 3 RESOLUTION RANGE LOW(ANGSTROMS): 47.74 REMARK 3 DATA CUTOFF (SIGMA(F)): 0.0 REMARK 3 DATACUTOFF HIGH (ABS(F)): 15276175.80 REMARK 3 DATA CUTOFF LOW (ABS(F)):0.000000 REMARK 3 COMPLETENESS (WORKING + TEST) (%): 99.5 REMARK 3NUMBER OF REFLECTIONS: 15190 REMARK 3 REMARK 3 FIT TO DATA USED INREFINEMENT. REMARK 3 CROSS-VALIDATION METHOD: THROUGHOUT REMARK 3 FREE RVALUE TEST SET SELECTION: RANDOM REMARK 3 R VALUE (WORKING SET): 0.215REMARK 3 FREE R VALUE: 0.269 REMARK 3 FREE R VALUE TEST SET SIZE (%):4.9 REMARK 3 FREE R VALUE TEST SET COUNT: 748 REMARK 3 ESTIMATED ERROROF FREE R VALUE: 0.010 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTIONBIN. REMARK 3 TOTAL NUMBER OF BINS USED: 6 REMARK 3 BIN RESOLUTION RANGEHIGH (A): 3.00 REMARK 3 BIN RESOLUTION RANGE LOW (A): 3.19 REMARK 3 BINCOMPLETENESS (WORKING + TEST) (%): 94.6 REMARK 3 REFLECTIONS IN BIN(WORKING SET): 2234 REMARK 3 BIN R VALUE (WORKING SET): 0.301 REMARK 3BIN FREE R VALUE: 0.350 REMARK 3 BIN FREE R VALUE TEST SET SIZE (%): 5.3REMARK 3 BIN FREE R VALUE TEST SET COUNT: 124 REMARK 3 ESTIMATED ERROROF BIN FREE R VALUE: 0.031 REMARK 3 REMARK 3 NUMBER OF NON-HYDROGENATOMS USED IN REFINEMENT. REMARK 3 PROTEIN ATOMS: 5007 REMARK 3 NUCLEICACID ATOMS: 0 REMARK 3 HETEROGEN ATOMS: 0 REMARK 3 SOLVENT ATOMS: 33REMARK 3 REMARK 3 B VALUES. REMARK 3 FROM WILSON PLOT (A**2): 54.9REMARK 3 MEAN B VALUE (OVERALL, A**2): 44.8 REMARK 3 OVERALL ANISOTROPICB VALUE. REMARK 3 B11 (A**2): 5.66 REMARK 3 B22 (A**2): 0.97 REMARK 3B33 (A**2): −6.63 REMARK 3 B12 (A**2): 0.00 REMARK 3 B13 (A**2): 0.00REMARK 3 B23 (A**2): 0.00 REMARK 3 REMARK 3 BULK SOLVENT MODELING.REMARK 3 METHOD USED: FLAT MODEL REMARK 3 KSOL: 0.313124 REMARK 3 BSOL:20.608 (A**2) REMARK 3 REMARK 3 ESTIMATED COORDINATE ERROR. REMARK 3 ESDFROM LUZZATI PLOT (A): 0.33 REMARK 3 ESD FROM SIGMAA (A): 0.39 REMARK 3LOW RESOLUTION CUTOFF (A): 5.00 REMARK 3 REMARK 3 CROSS-VALIDATEDESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM C-V LUZZATI PLOT (A): 0.44REMARK 3 ESD FROM C-V SIGMAA (A): 0.51 REMARK 3 REMARK 3 RMS DEVIATIONSFROM IDEAL VALUES. REMARK 3 BOND LENGTHS (A): 0.007 REMARK 3 BOND ANGLES(DEGREES): 1.0 REMARK 3 DIHEDRAL ANGLES (DEGREES): 22.1 REMARK 3IMPROPER ANGLES (DEGREES): 0.78 REMARK 3 REMARK 3 ISOTROPIC THERMALMODEL: RESTRAINED REMARK 3 REMARK 3 ISOTROPIC THERMAL FACTOR RESTRAINTS.RMS SIGMA REMARK 3 MAIN-CHAIN BOND (A**2): 1.46; 1.50 REMARK 3MAIN-CHAIN ANGLE (A**2): 2.62; 2.00 REMARK 3 SIDE-CHAIN BOND (A**2):1.63; 2.00 REMARK 3 SIDE-CHAIN ANGLE (A**2): 2.62; 2.50 REMARK 3 REMARK3 NCS MODEL: NONE REMARK 3 REMARK 3 NCS RESTRAINTS. RMS SIGMA/WEIGHTREMARK 3 GROUP 1 POSITIONAL (A): NULL; NULL REMARK 3 GROUP 1 B-FACTOR(A**2): NULL; NULL REMARK 3 REMARK 3 PARAMETER FILE 1: protein_rep.paramREMARK 3 PARAMETER FILE 2: water_rep.param REMARK 3 TOPOLOGY FILE 1:protein_no_ctertop REMARK 3 TOPOLOGY FILE 2: water.top REMARK 3 REMARK 3OTHER REFINEMENT REMARKS: NULL SSBOND 1 CYS L 23 CYS L 88 SSBOND 2 CYS H22 CYS H 96 SSBOND 3 CYS A 23 CYS A 88 SSBOND 4 CYS B 22 CYS B 96 SSBOND5 CYS C 94 CYS C 144 SSBOND 6 CYS C 99 CYS C 146 SSBOND 7 CYS D 94 CYS D144 SSBOND 8 CYS D 99 CYS D 146 CRYST1 184.306 55.813 70.991 90.00 90.0090.00 P 21 21 2 24 ORIGX1 1.000000 0.000000 0.000000 0.00000 ORIGX20.000000 1.000000 0.000000 0.00000 ORIGX3 0.000000 0.000000 1.0000000.00000 SCALE1 0.005426 0.000000 0.000000 0.00000 SCALE2 0.0000000.017917 0.000000 0.00000 SCALE3 0.000000 0.000000 0.014086 0.00000

FIG. 2 provides the three-dimensional structure of the XAB1 Fv complexwith human IL-17A, as obtained in Example 2. FIG. 2A shows the two XAB1Fv fragments in space-filling representation; the IL-17A homodimer isshown in cartoon representation. FIG. 2B shows the two XAB1 Fv fragmentsin cartoon representation; the IL-17A homodimer is shown inspace-filling representation. The heavy- and light-chain of the XAB1 Fvare represented as dark and light grey, respectively. One chain of theIL-17A homodimer is represented as light grey, the other is representedas dark grey.

A detailed analysis of the complex was performed. A careful visualinspection of the crystal structure with the programs Coot and Pymol wascarried out, and the amount of protein surface buried at theantibody-antigen interface was calculated with the program AREAIMOL ofthe CCP4 program suite. Intermolecular contacts were defined using acut-off distance of 3.9 Å between antibody and antigen atoms. Anoverview of binding can be summarized as follows. Binding of XAB1 issymmetrical; each Fv fragment binds to an equivalent epitope on theIL17A homodimer.

The binding of each Fv fragment buried on average 1732 Å² of combinedsurface, and involved 30 antibody and 25 IL-17A amino acid residues. Thecontribution to the buried surface of the XAB1 light-chain (around 560Å²) was greater than that of the heavy chain (around 275 Å²). Inaddition, CDRH2 did not make any direct contacts to IL-17A and appearedto be too far from the protein antigen to provide opportunities foraffinity maturation. The CDRH1 contribution appeared to be limited toone amino-acid side-chain only (Tyr32); this CDR was also too far fromIL-17A to offer opportunities for affinity maturation through amino acidsubstitutions. The XAB1 CDRH3 made multiple tight contacts with IL-17A.However, careful inspection of the structure in this region failed toreveal any opportunity for further enhancing these contacts by pointmutations; therefore, CDRH3 was deemed unsuitable as a target region foraffinity enhancement. In contrast, inspection of the light-chain CDRsshowed multiple opportunities for affinity maturation. Among the threelight-chain CDRs, CDRL1 was considered most promising, and based on thisobservation, the inventors proposed to randomize positions 30 to 32 ofthe light-chain in an attempt to strengthen contacts to IL-17A residuesArg124, Phe133 and Tyr85.

Affinity Maturation by Rational Design

Based on the results above, it was seen that the XAB1 interface withhomodimeric IL-17A was comparatively small and was characterized by adominant contribution from the light chain, no involvement from CDRH2,and mainly indirect contribution by CDRH1 (i.e. via stabilization ofCDRH3). Accordingly, the heavy chain of XAB1 did not appear to offerpromising opportunities for affinity maturation.

In contrast, the XAB1 light chain did offer some opportunities in aminoacid residues 30 to 32, with an optional insertion of up to 4 amino acidresidues (CDRL1), amino acids 51 to 53 and 56 (CDRL2) and amino acidresidues 92 and 93, with an optional insertion of up to 4 amino acidresidues.

The availability of the published crystal structure for homodimerichuman IL-17F, and the structure of homodimeric IL-17F in complex withthe human receptor IL-17RA allowed for predictions to be made based onthe observed structures of crystallized IL-17A and IL-17A in complexwith XAB1 (and variants thereof).

The structural similarities predicted between IL-17F and IL-17A (on thebasis of sequence identity and homology) were investigated. IL-17F andIL-17A bore a structural resemblance. The inventors hypothesized thatIL-17A would bind to the N-terminal domain of its receptor in the samemanner as has been shown for the published IL-17F/IL-17RA complex (Ely LK et al 2009, Nat Immunol. 10:1245-51).

On the basis of the observed structure and comparison of the knownsequences for human IL-17A and IL-17F, along with the sequence of IL-17Aderived from other species, a number of additional predictions were madeby the inventors:

It was expected that XAB1 (and antibodies variants derived there fromhaving an improved affinity for the epitope targeted by XAB1) would behighly specific for human IL-17A. It was hypothesized that suchantibodies would retain some cross-reactivity with IL-17A from otherspecies (on the basis of the high degree of conserved sequence identityor homology between species). However, on the basis of the availablesequence data and structural predictions it was not clear to what extentcross-reactivity with species variants of IL-17A could be expected.Given the lack of structural similarity with other Interleukins,cross-reactivity with such molecules (from humans or other species) wasexpected to be very unlikely.

In addition, differences between the sequences of IL-17A and IL-17F (inparticular N-terminal region) gave rise to predictions that theanti-IL-17A antibodies of the disclosure would not bind to IL-17F. Forexample, overlays of the two crystal structures indicated that sterichindrance would prevent binding between these antibodies and IL-17F.Furthermore, an extrapolation to the structure of IL-17AF heterodimersalso suggested that such interference, in particular in the N-terminalregion, would hamper binding of the antibodies to IL-17AF heterodimersand thereby result in a lack of binding to IL-17AF, i.e. a lack ofcross-reactivity by these antibodies for IL-17AF heterodimers.

Example 3. Generation of Affinity Matured Antibody Variants

Actual affinity maturation of the initial antibody XAB1 focused on thelight chain, for reasons discussed above. The work was carried out inthree steps: (i) library generation, (ii) library screening, and (iii)candidate characterisation.

The protein engineering work (i.e. affinity maturation) was carried outin the Fab fragment format for ease of handling. Candidates wereformatted back to full IgG after engineering.

(i) Library Generation

The DNA sequence encoding the variable domain of the light chain wasmutated to create a library of gene variants. Two different approaches(A and B) were used for library generation, providing two separatelibraries.

1) Method A—Random Mutation by Error Prone PCR:

The DNA region encoding the variable domain of the light chain of XAB1was randomly mutated using error prone PCR. In more detail, this regionwas amplified using the polymerase Mutazyme II, which introducedmutations at a high frequency (for more detail, see the guidancesupplied with the GeneMorph II random mutagenesis kit, supplied byStratagene #200550). However, any suitable random mutation technique orstrategy could be used.

The pool of PCR fragment variants was then cloned by cutting and pastinginto the expression vector of XAB1. Essentially, the parent, unmutatedsequence was cut out of the expression vector and replaced by a randomlymutagenized sequence which was pasted in its place. Standard molecularbiology techniques were used to accomplish this.

This resulted in a library of expression vector variants comprising avariety of randomly mutagenized variable domain sequences.

2) Method B—Mutation by Rational Design:

Under this approach, the generation of the library was guided by thestructural analysis carried out as a precursor to affinity maturation.Specific amino acid residues (in particular in CDR1 of the light chainof XAB1) were targeted based on the epitope and paratope informationderived from the crystal structure described above.

Three amino acid residues, selected on the basis of the crystalstructure information, were fully randomised. Standard molecular biologyapproaches were used for the construction.

Firstly, a fragment of the variable region encoding the appropriate CDRand a first part of the light chain framework was amplified by PCR,using degenerate oligonucleotides. That is, the oligonucleotides,encoding the CDR were synthesised in such a way as to provide a varietyof bases at a defined position or positions. Design of theoligonucleotide enabled randomization of specifically targeted aminoacid positions in the CDR by NNK degenerated codons (in which N standsfor all 4 bases, A, T, C and G and K for G and T) and allowed all 20natural amino acids at those positions.

Following this first step, a second fragment overlapping the first oneand encoding the remaining part of the light chain, was also amplifiedby PCR. Both fragments were then assembled by an “assembly” PCR togenerate the complete variable light chain and cloned back into theexpression vector in a ‘cut and paste’ manner. Thereby the parentalsequence was replaced with a range of rationally mutated sequences,whereby at specific amino acid positions all 20 natural amino acids wererepresented.

(ii) Library Screening

Once libraries comprising sequences encoding XAB1 variants had beengenerated it was necessary to screen them in order to select those whichhad superior characteristics to the parental XAB1 sequence, for examplehigher affinity for IL-17A.

Two screening techniques were used. Firstly, a high throughput screeningwas done by “colony filtration screening” (CFS). This assay permitted aconvenient screening of large number of clones. It allowed reduction topositive hits prior to ELISA screening, which was useful in particularfor the random approach “method A” as the library size was much larger(>10⁵) compared to the library size in “method B” (only 8000). ELISAscreening is convenient for 10⁴ clones or less and gives morequantitative results.

1) Colony Filtration Screening (CFS):

The protocol for CFS was based on Skerra et al. 1991, Anal Biochem196:151-155. Some adaptations were made.

E. coli colonies expressing the Fab variant libraries were grown on afilter on top of a Petri dish containing LB agar and glucose. Inparallel, a PVDF membrane was coated with the target protein (IL-17A).The coated membrane was placed on the agar plate. The filter withcolonies of Fab fragment expressing E. coli was placed on top of themembrane. The Fab fragments expressed by the cells diffused from thecolonies and bound the target IL-17A. The Fab fragment thus captured onthe PVDF membrane was then detected using a secondary antibodyconjugated with alkaline phosphatase for Western staining. Theconditions for selecting only variants with improved binding propertieswere previously established using XAB1 as reference.

More specifically, after transformation of E. coli cells with thelibrary, the cells were spread on a Durapore™ membrane filter (0.22 μmGV, Millipore®, cat # GVWP09050) placed on a Petri dish containing LBagar+1% glucose+antibiotic of interest. The plates were incubatedovernight at 30° C.

The PVDF membrane (Immobilon-P, Millipore®, cat # IPVH08100) was pre-wetin methanol, washed in PBS and coated with a huIL-17A solution at 1μg/ml in PBS. The membrane was incubated overnight at room temperature.After coating, the membrane was washed 2 times in Tris buffered saline(TBS)+0.05% Tween (TBST) and blocked two hours at room temperature in 5%milk TBST. Then, the membrane was washed four times in TBST and soakedin 2×YT medium with 1 mM IPTG. This membrane, called the capturemembrane was placed onto a LB agar plate with 1 mM IPTG+antibiotic ofinterest, and was covered with the Durapore membrane with the colonieson top. The resulting sandwich was incubated four hours at 30° C.

After this incubation, the capture membrane was washed 4 times with TBSTand blocked in 5% milk TBST for 1 hour at room temperature. Then, themembrane was washed once with TBST and incubated with a secondaryantibody (anti-hu_kappa light chain antibody, alkaline-phosphatase (AP)conjugated, Sigma # A3813, diluted 1:5000 in 2% milk TBST), 1 hour atroom temperature. Afterward, the membrane was washed 4 times in TBST,once in TBS and incubated in the substrate solution (SigmaFast BCIP/NBTtablet, 1 tablet in 10 ml H₂O). When the signal reached the expectedintensity the membrane was washed with water and allowed to dry.

After development of the signal on the capture membrane, the coloniesgiving stronger signal than the parental XAB1 were picked and allowed toproceed to a secondary ELISA screening described below.

2) ELISA Screening:

Following the CFS, ELISA was used to screen the candidates selected byCFS. In brief, for the relative low number of variants identified byerror-prone PCR mutagenesis (i.e. library A) the ELISA was performedmanually in a 96 well format. In contrast, for the libraries constructedby rational design (method B), a larger number of improved clones neededto be screened at ELISA level to be able to discriminate between theirdifferent binding affinity to IL-17A and identify the clones with thehighest affinity. An ELISA robot was used for that purpose in a 384 wellplate format. However, the ELISA protocol was the same in each case, theonly difference being the volumes of reagents.

a) Cell Cultures:

Clones were first grown overnight at 30° C., 900 rpm, in 2×YT medium+1%glucose+antibiotic of interest. The plates containing these cultureswere called master plates. The next day, aliquots of cultures from themaster plates were transferred into expression plates containing 2×YTmedium+0.1% glucose+antibiotic of interest. These plates were incubatedat 30° C., 900 rpm about 3 hours. Then isopropylβ-D-1-thiogalactopyranoside (IPTG) solution was added to a finalconcentration of 0.5 mM. The plates were incubated overnight at 30° C.,990 rpm.

The next day, lysis buffer (2×) Borate buffered saline (BBS) solution(Teknova # B0205)+2.5 mg/ml lysosyme+10 u/ml Benzonase) was added to thecultures. Plates were incubated 1 hour at room temperature, then 12.5%milk TBST was added for blocking. After 30 min incubation, cells lysateswere diluted 1:10 in 2% milk TBST and were transferred into the ELISAplates.

b) ELISA:

ELISA plates (Nunc Maxisorp) were coated with a huIL-17A solution at 1μg/ml during 1 hour. The plates were washed once with TBST and blocked 1hour with 5% milk TBST. After blocking, plates were washed 3 times withTBST and then, diluted cell lysates were loaded on the plates andincubated 1 hour. Afterward, plates were washed 3 times with TBST andwere incubated 1 hour with a secondary antibody AP conjugated.

The plates were finally washed 3 times with TBST and then incubated withthe substrate solution (AttoPhos substrate Set, Roche #11 681 982 001).The whole process was performed at room temperature.

In addition to the “classic” ELISA described above, modified ELISA werealso undertaken for a better discrimination between clones with veryhigh affinity (in the pico-molar range) for the target protein. An“off-rate” ELISA and a “competition” ELISA were developed for thispurpose, as detailed below.

c) “Off-Rate” ELISA:

For this assay, the modification compared to the “classic” ELISAprotocol regarded the washing step after the binding step (incubation ofcell lysate in ELISA plates). In the “classic” protocol, the plate waswashed 3 times with TBST. The washing solution was dispensed andimmediately aspirated, without any incubation time. For the “off-rate”ELISA, the plate was washed 6 times during at least 3 hours. This longwash increased the stringency of the assay, and allowed identifyingclones with a slow off-rate.

d) “Competition” ELISA:

This modified ELISA protocol included an extra step after the bindingstep. After incubation of cell lysate, the plates were washed 3 timeswith TBST and then, a solution of the parental XAB1 (200 nM in 2% milkTBST) was incubated overnight at room temperature. This long incubationwith an excess of the parental Fab allowed, as in the case of “off-rate”ELISA, to identify clones with slow off-rate, which lead to betterdiscrimination between clones with an affinity in the picomolar range.The rest of the protocol was similar to the “classic” ELISA protocol.The secondary antibody used in this case was an AP conjugated anti-Flagtag antibody, since the Fabs variants from the library had a Flag tag atthe C-terminus of the heavy chain but not the parental XAB1 Fab used forthe competition.

(iii) Candidate Characterisation

The hits identified during the screening were produced on a larger scalefor further physicochemical characterisation and to confirm highaffinity binding to IL-17A, and/or other advantageous properties inadditional assays. These are described below in more detail.

(iv) Results: Screening and Initial Characterization of CandidatesFollowing Affinity Maturation of XAB1

1) Random Mutagenesis Approach (Method A):

The mutation rate after the error-prone PCR library generation was foundto peak at 2 to 3 mutations per gene. Around 3×10⁴ clones were screenedby colony filter screening and a number of 94 clones were identified asimproved and allowed to proceed to binding, off-rate and competitionELISA. ELISA data in combination with sequencing results led to theidentification of 6 candidates highlighting 3 potential hot spots forimprovement, Gly at position 28 to Val (G28V) in LCDR1, Gly at position66 to Val (G66V) or Ser (G66S) in framework 3; Asn92 to Asp (N92D) inLCDR3 (data not shown, but positioning is identical to that of XAB2,V_(L), i.e. SEQ ID NO: 25).

A stop codon was observed in one of the clones, but was not relevant asthe E. coli strain used was an amber suppressor strain allowingread-through. Based on the data obtained, a G28V and G66V mutationappeared to cause the best improvement. A variant of XAB1 was generatedby standard molecular biology techniques carrying the two pointmutations mentioned. A further variant was cloned having the N92Dsubstitution in addition, in order to test whether the removal of thepotential post-translational deamidation site (N92, S93) would bebeneficial. More detailed profiling was done on those two variants, inparticular of the triple mutant variant referred to as XAB_A2 whichfinally led to XAB2. In XAB2, amino acids number 1 to 23 according tothe Kabat definition constitute framework 1, amino acids number 24 to 34(Kabat) constitute LCDR1, amino acids number 35 to 49 (Kabat) constituteframework 2, amino acids 50 to 56 (Kabat) constitute LCDR2, amino acids57 to 88 (Kabat) constitute framework 3, amino acids 89 to 97 (Kabat)constitute LCDR3 and amino acids 98 to 107 (Kabat) constitute framework4. The same subdivision of other VL sequences according to embodimentsof the disclosure also applies.

Thus, the G66V substitution mentioned above is in a framework region,which is called the outer loop. This framework region is able tocontribute to binding in some cases. Based on the available structuralinformation it was retrospectively suggested that this mutation indeedmight be able to interact with a region of IL-17A which cannot beresolved from the crystal structure but may be in proximity to the outerloop.

2) Rational Mutagenesis Approach (Method B):

A snapshot of the amino acid distribution at the randomized positionswas generated by sequencing of 32 randomly picked members. There was nosignificant bias, though statistics with this low number of sequencescannot be done. Around 4×10⁴ clones were screened which oversampled thetheoretical library size of 8000. A high number of hits were identifiedand 2630 clones were allowed to proceed to ELISA screening. Performingbinding, off-rate and competition ELISA, 60 clones with the highestimprovements were sequenced. In those 60 clones 22 unique sequences werefound, and the result is summarized in Table 11.

TABLE 11 ELISA of all selected 22 unique candidates. Values arenormalized to parental Fab XAB1. The representation indicates how oftena certain sequence was found within the 60 hits. The difference in aminoacid sequence is given in the three last columns. XAB1 has the aminoacids I S A at those positions. ELISA signals determined from crudeextract of Fab expression culture from E. coli. Off- Candidate Classicrate Competition Represen- name ELISA ELISA ELISA tation % 30 31 32MB491 2.1 43.0 44.2 5 F F W MB483 3.1 47.1 45.2 2 F W T MB447 3.0 45.557.0 5 F W W MB457 2.7 34.7 41.0 5 I W S MB464 2.7 34.9 36.9 7 I Y QMB432 2.3 44.2 37.3 12 L F A MB454 2.9 34.2 36.6 2 L W A MB444 3.2 48.952.4 2 L W E MB456 2.4 45.1 46.7 2 L W H MB440 2.8 52.5 54.0 5 L W QMB450 2.9 41.5 53.3 5 M W W MB435 2.7 44.7 44.6 2 N W E MB438 2.7 41.541.1 7 P Y A MB453 2.7 43.3 46.4 9 V F W MB448 2.9 40.4 51.5 5 V W MMB486 1.9 58.5 64.9 2 W W M MB434 2.4 44.4 39.5 7 W W Y MB458 2.7 33.042.1 5 W Y Q MB463 2.7 34.2 31.6 2 Y F E MB468 2.8 43.9 60.0 5 Y W EMB433 2.3 39.7 29.3 2 Y W G MB461 2.9 49.8 62.8 2 Y W T

Of the 22 unique clones, 6 were selected for 0.5 L scale standard E.coli expression and two step purification by IMAC (Ni-NTA) and SEC.Purified Fabs were then used to confirm the improvement in binding byELISA.

ELISA results of selected and purified Fab candidates in comparison toXAB1 are shown in FIG. 3, where the graph numbering corresponds to thecandidate designation as follows: 1 is MB440; 2 is MB464; 3 is MB468; 4is MB444; 5 is MB435; 6 is MB463; 7 is XAB1.

FIG. 3A is a graph showing the normalized signal versus the Fabconcentration (M). It can be seen that all the selected clones resultedin a higher signal than XAB1. FIG. 3B is a graph showing the normalizedremaining signal versus the washing incubation time (hours). All theselected clones result in a higher signal than XAB1. FIG. 3C is a graphshowing the normalized signal versus the Fab competitor concentration(M). Again, it can be seen that all the selected clones result in ahigher signal than XAB1.

Example 4. Targeting a Potential Post-Translational Deamidation Site

The inventors hypothesized that the amino acid motif asparagine followedby glycine (NG) or, to lower extend also when followed by serine (NS),may be susceptible to post-translational deamidation. Such motifs arepresent in L-CDR2 (position 56/57) and L-CDR3 (92/93) of the antibodyXAB1. Four IgG variants were generated in order to test whether the NGsite could be removed without affecting binding and activity properties.These four point mutation variants were cloned by standard molecularbiology procedures and produced by standard transient transfection ofHEK cells in 100 ml scale and purified via a protein A column.

Purified IgG variants were analyzed in an in vitro neutralization assay(e.g. as described in examples 12 and 13) to compare their activity tothe parental XAB1 IgG. Results showed that out of these four variants,three had a reduced activity. But the candidate XAB_B12 (mutation N56Q)retained activity compared to the parental XAB1.

TABLE 12 Overview of sequence modifications to XAB1, and correspondingeffect on in vitro neutralization. Kabat CDR L-CDR2 Residue# IC50(nM) 4950 51 52 53 54 55 56 57 Hu Hu IgG Generic Kabat# IL-6 IL-8 variants name49 50 51 52 53 54 55 56 57 sec sec XAB1 XAB1 Y D A S S L E N G 4 3XAB_G57T XAB_A6 Y D A S S L E N T 22 23 XAB_N56Q XAB_B12 Y D A S S L E QG 2 3 XAB_N56T XAB_B13 Y D A S S L E T G 11 15 XAB_N56S XAB_B14 Y D A SS L E S G 13 17

Having thus identified the most suitable substitution, it was introducedto the most promising hits identified during the affinity maturationprocess, resulting in XAB2 (XAB_A2 N56Q), XAB3 (MB468 N56Q), XAB4 (MB435N56Q). They were produced by standard transient transfection of HEKcells and purified via protein A column along with XAB5 (MB435), whichstill carried the NG site.

The NG motif was removed (N56Q) for the XAB2, XAB3, XAB4, but was stillpresent in XAB5. The NS motif in L-CDR3 was removed (N92D) in XAB2, asfound during the random affinity maturation approach. Therefore, anoptimal set of variants was available to test the susceptibility fordeamidation of the potential sites.

The four purified candidates were diluted in a buffer pH 8 and incubatedat 40° C. in order to force the deamidation reaction. Aliquots weretaken at several time points to determine the degree of deamidation bycation exchange chromatography (CEX), according to principles well knownto a person skilled in the art, and the in vitro neutralization activityby a cell based assay was determined (e.g. as described in examples 12and 13).

CEX results showed an increase of acidic variants percentage over time,as expected for any IgG, likely due to post-translational modificationsites in the antibody framework, but the extent of increase was higherfor XAB5 than for the other candidates, i.e. 72% vs. 46% after one weekand 94% vs. 83% after 4 weeks. Finally, in vitro neutralization activityassay results correlated with the CEX results, showing that XAB5 lostactivity after 4 weeks incubation during forced deamidation condition.Size-exclusion chromatography-multi angle light scattering methodology(SEC-MALS), well known to a person skilled in the art, was used tomonitor the aggregation levels in the samples.

The data is summarized in Table 13.

TABLE 13 Analysis by SEC-MALS, in vitro neutralization activity and CEX.M ^(a)) EC₅₀ ^(b)) CEX^(c)) M ^(a)) EC₅₀ ^(b)) CEX^(c)) [%] [ng/ml] [%][%] [ng/ml] [%] Antibody T = 0 weeks T = 1 weeks XAB2 99 45 15 98 n.d.45 XAB3 99 40 14 98 n.d. 44 XAB5 99 45 18 98 n.d. 72 XAB4 99 48 15 98n.d. 48 M ^(a)) EC₅₀ ^(b)) CEX^(c)) [%] [ng/ml] [%] NG^(d)) NS^(d))Antibody T = 4 weeks sites sites XAB2 95 47 85 0 0 XAB3 97 40 81 0 1XAB5 94 61 94 1 1 XAB4 94 47 84 0 1 ^(a)) monomer by SEC-MALS, ^(b))inhibition of IL-6 secretion after cell stimulation with 80 ng/ml IL-17,^(c))acidic variants by exchange chromatography, ^(d))number of sites inCDRs (framework region not considered)

These data indicated successful removal of a potentialpost-translational deamidation site, which could have had an effect onantibody activity. This is advantageous, since XAB2, XAB3 and XAB4 aretherefore likely to achieve a more homogeneous product than XAB1 as nopost-translational deamidation can occur during production or storageaffecting the antibody activity.

Example 5. X-Ray Analysis of Antibody Variants Derived by AffinityMaturation: XAB2

In brief, the XAB2 Fv was cloned and expressed in E. coli TGf1− with aC-terminal hexahistidine tag on the heavy-chain and a C-terminalStrep-tag on the light-chain, according to principles well known to aperson skilled in the art. The recombinant protein was purified byNi-chelate chromatography and size-exclusion chromatography (SPX-75).

The XAB2 Fv fragment complex with human IL-17A was then prepared usingstandard methodology. In brief, human IL-17A (1.5 mg) was mixed with anexcess of XAB2 Fv (3.7 mg) and the complex was run on a S100size-exclusion chromatography, in 10 mM TRIS pH 7.4, 25 mM NaCl. Theprotein complex was then concentrated by ultra-filtration to 26.3 mg/mland crystallized.

Standard crystallization protocols were followed. In brief, crystalswere grown at 19° C. in SD2 96—well plates, using the method of vapourdiffusion in sitting drops. The protein stock was mixed 1:1 with acrystallization buffer containing 0.2M calcium acetate, 20% PEG 3,350.Total drop size was 0.4 μl. Prior to X-ray data collection, one crystalwas briefly transferred into a 1:1 mix of the crystallization bufferwith 30% PEG 3,350, 30% glycerol, and then flash cooled into liquidnitrogen.

X-ray data collection and processing was carried out using standardprotocols. Briefly, X-ray data to 2.0 Å resolution were collected at theSwiss Light Source, beamline X06DA, with a MAR225 CCD detector, using1.0000 Å X-ray radiation. In total, 360 images of 0.5° oscillation eachwere recorded at a crystal-to-detector distance of 190 mm and processedwith the XDS software package. The crystal belonged to space groupP2₁2₁2 with cell parameters a=184.72 Å, b=55.56 Å, c=71.11 Å, α=β=γ=90°.R-sym to 2.0 Å resolution was 5.2% and data completeness 100.0%.

As the crystal of the XAB2 Fv complex was highly isomorphous with thecrystal of the XAB1 Fv complex (Example 2), the structure of the latterwas used as input model for an initial run of crystallographicrefinement with the program CNX. Iterative model correction andrefinement was performed with Coot (Crystallographic Object-OrientedToolkit) and CNX (Crystallography & NMR eXplorer) version 2002, until nofurther significant improvements could be made to the crystallographicmodel. Final R- and R-free for all data were 0.214 and 0.259,respectively. The final refined model showed a root-mean-squaredeviation (RMSD) from ideal bond lengths and bond angles of 0.005 Å and0.9°, respectively.

Results

The results of the X-ray refinement of the XAB2 Fv complex with humanIL-17A are provided in Table 14 and the three-dimensional structure ofthis complex is shown in FIG. 4. The X-ray crystallography analysisconfirmed that the variant antibody XAB2 retained the target specificityand bound with high affinity to essentially the same epitope as theparental XAB1 antibody. However, in the XAB1 complex structure, thelight-chain loop comprising Gly66 adopts a conformation that is nolonger possible when this residue is mutated to a valine. As aconsequence, in the XAB2 complex, the Gly66 to valine mutation (G66V)forces the loop to adopt a new conformation, and the valine side-chainmakes hydrophobic contacts to Ile51 of IL-17A (FIG. 5). Two more IL-17Aresidues, Pro42 and Arg43, become visible (ordered) in this crystalstructure. These antigen residues make additional binding interactionswith the XAB2 antibody, in particular hydrophobic contacts to Va128(FIG. 5).

TABLE 14 X-ray refinement of the XAB2 Fv complex with IL-17A obtained bythe program CNX. REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM: CNX2002 REMARK 3 AUTHORS: Brunger, Adams, Clore, Delano, REMARK 3     Gros,Grosse-Kunstleve, Jiang, REMARK 3     Kuszewski, Nilges, Pannu, Read,REMARK 3     Rice, Simonson, Warren REMARK 3     and REMARK 3    Accelrys Inc., REMARK 3     (Badger, Berard, Kumar, Szalma, REMARK 3    Yip, Dzakula). REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3RESOLUTION RANGE HIGH (ANGSTROMS): 2.00 REMARK 3 RESOLUTION RANGE LOW(ANGSTROMS): 71.11 REMARK 3 DATA CUTOFF (SIGMA(F)): 0.0 REMARK 3 DATACUTOFF HIGH (ABS(F)): 2329350.20 REMARK 3 DATA CUTOFF LOW (ABS(F)):0.000000 REMARK 3 COMPLETENESS (WORKING + TEST) (%): 99.8 REMARK 3NUMBER OF REFLECTIONS: 50409 REMARK 3 REMARK 3 FIT TO DATA USED INREFINEMENT. REMARK 3 CROSS-VALIDATION METHOD: THROUGHOUT REMARK 3 FREE RVALUE TEST SET SELECTION: RANDOM REMARK 3 R VALUE (WORKING SET): 0.214REMARK 3 FREE R VALUE: 0.259 REMARK 3 FREE R VALUE TEST SET SIZE (%):5.0 REMARK 3 FREE R VALUE TEST SET COUNT: 2521 REMARK 3 ESTIMATED ERROROF FREE R VALUE: 0.005 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTIONBIN. REMARK 3 TOTAL NUMBER OF BINS USED: 6 REMARK 3 BIN RESOLUTION RANGEHIGH (A): 2.00 REMARK 3 BIN RESOLUTION RANGE LOW (A): 2.13 REMARK 3 BINCOMPLETENESS (WORKING + TEST) (%): 100.0 REMARK 3 REFLECTIONS IN BIN(WORKING SET): 7858 REMARK 3 BIN R VALUE (WORKING SET): 0.262 REMARK 3BIN FREE R VALUE: 0.304 REMARK 3 BIN FREE R VALUE TEST SET SIZE (%): 5.0REMARK 3 BIN FREE R VALUE TEST SET COUNT: 414 REMARK 3 ESTIMATED ERROROF BIN FREE R VALUE: 0.015 REMARK 3 REMARK 3 NUMBER OF NON-HYDROGENATOMS USED IN REFINEMENT. REMARK 3 PROTEIN ATOMS: 5055 REMARK 3 NUCLEICACID ATOMS: 0 REMARK 3 HETEROGEN ATOMS: 0 REMARK 3 SOLVENT ATOMS: 376REMARK 3 REMARK 3 B VALUES. REMARK 3 FROM WILSON PLOT (A**2): 27.8REMARK 3 MEAN B VALUE (OVERALL, A**2): 37.3 REMARK 3 OVERALL ANISOTROPICB VALUE. REMARK 3 B11 (A**2): −0.85 REMARK 3 B22 (A**2): 3.93 REMARK 3B33 (A**2): −3.08 REMARK 3 B12 (A**2): 0.00 REMARK 3 B13 (A**2): 0.00REMARK 3 B23 (A**2): 0.00 REMARK 3 REMARK 3 BULK SOLVENT MODELING.REMARK 3 METHOD USED: FLAT MODEL REMARK 3 KSOL: 0.338594 REMARK 3 BSOL:46.0594 (A**2) REMARK 3 REMARK 3 ESTIMATED COORDINATE ERROR. REMARK 3ESD FROM LUZZATI PLOT (A): 0.25 REMARK 3 ESD FROM SIGMAA (A): 0.19REMARK 3 LOW RESOLUTION CUTOFF (A): 5.00 REMARK 3 REMARK 3CROSS-VALIDATED ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM C-VLUZZATI PLOT (A): 0.31 REMARK 3 ESD FROM C-V SIGMAA (A): 0.22 REMARK 3REMARK 3 RMS DEVIATIONS FROM IDEAL VALUES. REMARK 3 BOND LENGTHS (A):0.005 REMARK 3 BOND ANGLES (DEGREES): 0.9 REMARK 3 DIHEDRAL ANGLES(DEGREES): 21.0 REMARK 3 IMPROPER ANGLES (DEGREES): 0.70 REMARK 3 REMARK3 ISOTROPIC THERMAL MODEL: RESTRAINED REMARK 3 REMARK 3 ISOTROPICTHERMAL FACTOR RESTRAINTS. RMS SIGMA REMARK 3 MAIN-CHAIN BOND (A**2):1.49; 1.50 REMARK 3 MAIN-CHAIN ANGLE (A**2): 2.44; 2.00 REMARK 3SIDE-CHAIN BOND (A**2): 1.95; 2.00 REMARK 3 SIDE-CHAIN ANGLE (A**2):2.93; 2.50 REMARK 3 REMARK 3 NCS MODEL: NONE REMARK 3 REMARK 3 NCSRESTRAINTS. RMS SIGMA/WEIGHT REMARK 3 GROUP 1 POSITIONAL (A): NULL ;NULL REMARK 3 GROUP 1 B-FACTOR (A**2): NULL ; NULL REMARK 3 REMARK 3PARAMETER FILE 1: protein_rep.param REMARK 3 PARAMETER FILE 2:water_rep.param REMARK 3 PARAMETER FILE 3: ion.param REMARK 3 TOPOLOGYFILE 1: protein.top REMARK 3 TOPOLOGY FILE 2: water.top REMARK 3TOPOLOGY FILE 4: ion.top REMARK 3 REMARK 3 OTHER REFINEMENT REMARKS:NULL SSBOND 1 CYS L 23 CYS L 88 SSBOND 2 CYS H 22 CYS H 96 SSBOND 3 CYSA 23 CYS A 88 SSBOND 4 CYS B 22 CYS B 96 SSBOND 5 CYS C 94 CYS C 144SSBOND 6 CYS C 99 CYS C 146 SSBOND 7 CYS D 94 CYS D 144 SSBOND 8 CYS D99 CYS D 146 CRYST1 184.719 55.558 71.109 90.00 90.00 90.00 P 21 21 2 24ORIGX1 1.000000 0.000000 0.000000 0.00000 ORIGX2 0.000000 1.0000000.000000 0.00000 ORIGX3 0.000000 0.000000 1.000000 0.00000 SCALE10.005414 0.000000 0.000000 0.00000 SCALE2 0.000000 0.017999 0.0000000.00000 SCALE3 0.000000 0.000000 0.014063 0.00000

FIG. 4 provides the three-dimensional structure of the XAB2 Fv complexwith human IL-17A. FIG. 4A shows the two XAB2 Fv fragments inspace-filling representation, and the IL-17A homodimer is shown incartoon representation. FIG. 4B shows the two XAB2 Fv fragments incartoon representation, and the IL-17A homodimer is shown inspace-filling representation. The heavy- and light-chain of the XAB2 Fvare shown in dark and light grey, respectively. One chain of the IL-17Ahomodimer is shown in light grey, the other is shown in dark grey.

FIG. 5 provides the three-dimensional structure of the XAB2 Fv complexwith human IL-17A as a close-up view of the antibody L-CDR1 and outerloop regions, bearing the glycine to valine mutations (G28V and G66V,respectively). The G66V mutation leads to a change in the conformationof the outer loop, as well as to additional antibody-antigen contacts toIL-17A residues Pro42, Arg43 and Ile51. The XAB2 Fv is represented inlight-grey cartoon, and the human IL-17A homodimer in darker shades ofgrey. Ile51 does not belong to the same IL-17A subunit as Pro42 andArg43.

Example 6. X-Ray Analysis of Antibody Variants Derived by AffinityMaturation: XAB5

The XAB5 Fv was cloned and expressed in E. coli TGf1- with a C-terminalhexahistidine tag on the heavy-chain and a C-terminal Strep-tag on thelight-chain. The recombinant protein was purified by Ni-chelatechromatography followed by size-exclusion chromatography on a SPX-75column, in PBS buffer. LC-MS analysis showed the expected mass for theheavy-chain (13703.4 Da), and the presence of two forms of thelight-chain: full-length (115aa, 12627.3 Da, ca. 27%) and with truncatedStrep-tag (A1 to Q112; 12222.8 Da; ca. 73%).

The XAB5 Fv fragment complex with human IL-17A was then prepared usingstandard methodology. In brief, human IL-17A (1.4 mg) was mixed with anexcess of XAB5 Fv (3.4 mg) and the complex was run on a S100size-exclusion chromatography, in 10 mM TRIS pH 7.4, 25 mM NaCl. Theprotein complex was then concentrated by ultra-filtration to 16.5 mg/mland crystallized.

Standard crystallization protocols were followed. In brief, crystalswere grown at 19° C. in SD2 96 well-plates, using the method of vapourdiffusion in sitting drops. The protein stock was mixed 1:1 with acrystallization buffer containing 15% PEG 5,000 MME, 0.1M MES pH 6.5,0.2M ammonium sulfate. Total drop size was 0.4 μl. Prior to X-ray datacollection, one crystal was briefly transferred into a 1:1 mix of thecrystallization buffer with 20% PEG 5,000 MME, 40% glycerol, and thenflash cooled into liquid nitrogen.

X-ray data collection and processing was carried out using standardprotocols. Briefly, X-ray data to 3.1 Å resolution were collected at theSwiss Light Source, beamline X10SA, with a Pilatus detector, using1.00000 Å X-ray radiation. In total, 720 images of 0.25° oscillationeach were recorded at a crystal-to-detector distance of 520 mm andprocessed with the XDS software package. The crystal belonged to spacegroup C222₁ with cell parameters a=55.37 Å, b=84.08 Å, c=156.35 Å,α=β=γ=90°. R-sym to 3.1 Å resolution was 8.9% and data completeness99.7%.

The structure was determined by molecular replacement with the programPhaser, using search models derived from the previously-determined XAB2Fv complex. Iterative model correction and refinement was performed withCoot (Crystallographic Object-Oriented Toolkit) and CNX (Crystallography& NMR eXplorer) version 2002, until no further significant improvementscould be made to the crystallographic model Final R- and R-free for alldata were 0.222 and 0.305, respectively. The final refined model showeda root-mean-square deviation (RMSD) from ideal bond lengths and bondangles of 0.008 Å and 1.2°, respectively.

Results

The results of the X-ray refinement of the XAB5 Fv complex with humanIL-17A are provided in Table 15 and the three-dimensional structure ofthis complex is shown in FIG. 6. In this crystal structure, the XAB5 Fvcomplex has exact crystallographic 2-fold symmetry: the asymmetric unitof the crystal contains only one half of the whole, dimeric complex. TheXAB5 Fv makes contacts to both IL-17A subunits, but the vast majority ofthe intermolecular contacts are to only one subunit (around 90% of theIL-17A surface buried by the XAB5 Fv is contributed by one IL-17Asubunit). The X-ray crystallography analysis confirmed that the variantantibody XAB5 retained the target specificity and bound with highaffinity to essentially the same epitope as the parental XAB1 antibody.However, in the XAB5 complex structure, the light-chain CDRL1 bearsthree point mutations which provide enhanced binding to human IL-17A.Trp 31 of the XAB5 light-chain is engaged in strong hydrophobic/aromaticinteractions with Tyr 85 of IL-17A and, to a lesser extent, Phe 133 ofIL-17A. Asn 30 of the XAB5 light-chain donates a H-bond to themain-chain carbonyl of Pro 130 of IL-17A and is in van der Waals contactto Leu 49 (same IL-17A subunit) and Val 45 (other IL-17A subunit). Glu32 of the XAB5 light-chain stabilizes the CDRL1 loop throughintramolecular H-bonded interactions. Furthermore, Glu 32 makesfavorable electrostatic interactions with Arg 124 of IL-17A, but is notengaged into a “head-to-head” salt-bridge interaction (FIG. 7).

TABLE 15 X-ray refinement of the XAB5 Fv complex with IL-17A obtained bythe program CNX. REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM: CNX2002 REMARK 3 AUTHORS: Brunger, Adams, Clore, Delano, REMARK 3     Gros,Grosse-Kunstleve, Jiang, REMARK 3     Kuszewski, Nilges, Pannu, Read,REMARK 3     Rice, Simonson, Warren REMARK 3     and REMARK 3    Accelrys Inc., REMARK 3     (Badger, Berard, Kumar, Szalma, REMARK 3    Yip, Dzakula). REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3RESOLUTION RANGE HIGH (ANGSTROMS): 3.11 REMARK 3 RESOLUTION RANGE LOW(ANGSTROMS): 46.25 REMARK 3 DATA CUTOFF (SIGMA(F)): 0.0 REMARK 3 DATACUTOFF HIGH (ABS(F)): 3778977.84 REMARK 3 DATA CUTOFF LOW (ABS(F)):0.000000 REMARK 3 COMPLETENESS (WORKING + TEST) (%): 99.0 REMARK 3NUMBER OF REFLECTIONS: 6801 REMARK 3 REMARK 3 FIT TO DATA USED INREFINEMENT. REMARK 3 CROSS-VALIDATION METHOD: THROUGHOUT REMARK 3 FREE RVALUE TEST SET SELECTION: RANDOM REMARK 3 R VALUE (WORKING SET): 0.222REMARK 3 FREE R VALUE: 0.305 REMARK 3 FREE R VALUE TEST SET SIZE (%):5.0 REMARK 3 FREE R VALUE TEST SET COUNT: 340 REMARK 3 ESTIMATED ERROROF FREE R VALUE: 0.017 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTIONBIN. REMARK 3 TOTAL NUMBER OF BINS USED: 6 REMARK 3 BIN RESOLUTION RANGEHIGH (A): 3.10 REMARK 3 BIN RESOLUTION RANGE LOW (A): 3.29 REMARK 3 BINCOMPLETENESS (WORKING + TEST) (%): 89.9 REMARK 3 REFLECTIONS IN BIN(WORKING SET): 961 REMARK 3 BIN R VALUE (WORKING SET): 0.293 REMARK 3BIN FREE R VALUE: 0.403 REMARK 3 BIN FREE R VALUE TEST SET SIZE (%): 4.9REMARK 3 BIN FREE R VALUE TEST SET COUNT: 50 REMARK 3 ESTIMATED ERROR OFBIN FREE R VALUE: 0.057 REMARK 3 REMARK 3 NUMBER OF NON-HYDROGEN ATOMSUSED IN REFINEMENT. REMARK 3 PROTEIN ATOMS: 2492 REMARK 3 NUCLEIC ACIDATOMS: 0 REMARK 3 HETEROGEN ATOMS: 5 REMARK 3 SOLVENT ATOMS: 4 REMARK 3REMARK 3 B VALUES. REMARK 3 FROM WILSON PLOT (A**2): 85.2 REMARK 3 MEANB VALUE (OVERALL, A**2): 71.0 REMARK 3 OVERALL ANISOTROPIC B VALUE.REMARK 3 B11 (A**2): 23.21 REMARK 3 B22 (A**2): 7.23 REMARK 3 B33(A**2): −30.44 REMARK 3 B12 (A**2): 0.00 REMARK 3 B13 (A**2): 0.00REMARK 3 B23 (A**2): 0.00 REMARK 3 REMARK 3 BULK SOLVENT MODELING.REMARK 3 METHOD USED: FLAT MODEL REMARK 3 KSOL: 0.389339 REMARK 3 BSOL:59.5295 (A**2) REMARK 3 REMARK 3 ESTIMATED COORDINATE ERROR. REMARK 3ESD FROM LUZZATI PLOT (A): 0.35 REMARK 3 ESD FROM SIGMAA (A): 0.42REMARK 3 LOW RESOLUTION CUTOFF (A): 5.00 REMARK 3 REMARK 3CROSS-VALIDATED ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM C-VLUZZATI PLOT (A): 0.51 REMARK 3 ESD FROM C-V SIGMAA (A): 0.45 REMARK 3REMARK 3 RMS DEVIATIONS FROM IDEAL VALUES. REMARK 3 BOND LENGTHS (A):0.008 REMARK 3 BOND ANGLES (DEGREES): 1.2 REMARK 3 DIHEDRAL ANGLES(DEGREES): 23.1 REMARK 3 IMPROPER ANGLES (DEGREES): 0.73 REMARK 3 REMARK3 ISOTROPIC THERMAL MODEL: RESTRAINED REMARK 3 REMARK 3 ISOTROPICTHERMAL FACTOR RESTRAINTS. RMS SIGMA REMARK 3 MAIN-CHAIN BOND (A**2):1.40; 1.50 REMARK 3 MAIN-CHAIN ANGLE (A**2): 2.49; 2.00 REMARK 3SIDE-CHAIN BOND (A**2): 1.82; 2.00 REMARK 3 SIDE-CHAIN ANGLE (A**2):2.93; 2.50 REMARK 3 REMARK 3 NCS MODEL: NONE REMARK 3 REMARK 3 NCSRESTRAINTS. RMS SIGMA/WEIGHT REMARK 3 GROUP 1 POSITIONAL (A): NULL; NULLREMARK 3 GROUP 1 B-FACTOR (A**2): NULL; NULL REMARK 3 REMARK 3 PARAMETERFILE 1: protein_rep.param REMARK 3 PARAMETER FILE 2: water_rep.paramREMARK 3 PARAMETER FILE 3: ion.param REMARK 3 TOPOLOGY FILE 1:protein_no_ctertop REMARK 3 TOPOLOGY FILE 2: water.top REMARK 3 TOPOLOGYFILE 4: ion.top REMARK 3 REMARK 3 OTHER REFINEMENT REMARKS: NULL SSBOND1 CYS S 23 CYS S 88 SSBOND 2 CYS S 22 CYS S 96 SSBOND 3 CYS S 94 CYS S144 SSBOND 4 CYS S 99 CYS S 146 CRYST1 55.372 84.082 156.350 90.00 90.0090.00 C 2 2 21  24 ORIGX1 1.000000 0.000000 0.000000 0.00000 ORIGX20.000000 1.000000 0.000000 0.00000 ORIGX3 0.000000 0.000000 1.0000000.00000 SCALE1 0.018060 0.000000 0.000000 0.00000 SCALE2 0.0000000.011893 0.000000 0.00000 SCALE3 0.000000 0.000000 0.006396 0.00000

FIG. 6 provides the three-dimensional structure of the XAB5 Fv complexwith human IL-17A. The full homodimeric complex with exactcrystallographic two-fold symmetry is shown here. FIG. 6A shows the twoXAB5 Fv fragments in space-filling representation, and the IL-17Ahomodimer is shown in cartoon representation. FIG. 6B shows the two XAB5Fv fragments in cartoon representation, and the IL-17A homodimer isshown in space-filling representation. The heavy- and light-chain of theXAB5 Fv are shown in dark and light grey, respectively. One chain of theIL-17A homodimer is shown in light grey, the other is shown in darkgrey.

FIG. 7 provides the three-dimensional structure of the XAB5 Fv complexwith human IL-17A. Close-up view of the antibody L-CDR1 bearing thethree mutations found by the structure-guided biased library approach:Asn 30, Trp 31 and Glu 32. These XAB5 side-chains contribute new bindinginteractions to the antigen human IL-17A, in particular to

IL-17A residues Tyr85, Phe133, Arg124, Pro 130, Leu 49 (all from thesame IL-17A subunit) and Val 45 (from the other IL-17A subunit).

Example 7. X-Ray Analysis of Antibody Variants Derived by AffinityMaturation: XAB4

The XAB4 Fv was cloned and expressed in E. coli TG1 cells with aC-terminal hexahistidine tag on the heavy-chain and a C-terminalStrep-tag on the light-chain. The recombinant protein was purified byNi-chelate chromatography.

The XAB4 Fv fragment complex with human IL-17A was then prepared usingstandard methodology. In brief, human IL-17A (0.5 mg) was mixed with anexcess of XAB4 Fv (1.2 mg) and the complex was run on a SPX-75size-exclusion chromatography, in 10 mM TRIS pH 7.4, 25 mM NaCl. Theprotein complex was then concentrated by ultra-filtration to 6.9 mg/mland crystallized.

Standard crystallization protocols were followed. In brief, crystalswere grown at 19° C. in VDX 24 well-plates, using the method of vapourdiffusion in hanging drops. The protein stock was mixed 2:1 with acrystallization buffer containing 15% PEG 5,000 MME, 0.1M MES pH 6.5,0.2M ammonium sulfate. Total drop size was 3.0 μl. Prior to X-ray datacollection, one crystal was briefly transferred into a 1:1 mix of thecrystallization buffer with 25% PEG 5,000 MME, 20% glycerol, and thenflash cooled into liquid nitrogen.

X-ray data collection and processing was carried out using standardprotocols. Briefly, X-ray data to 3.15 Å resolution were collected atthe Swiss Light Source, beamline X10SA, with a Pilatus detector, using0.99984 Å X-ray radiation. In total, 720 images of 0.25° oscillationeach were recorded at a crystal-to-detector distance of 500 mm andprocessed with the XDS software package. The crystal belonged to spacegroup C222₁ with cell parameters a=55.76 Å, b=87.11 Å, c=156.31 Å,α=β=γ=90°. R-sym to 3.15 Å resolution was 5.5% and data completeness99.9%.

As the crystal of the XAB4 Fv complex was nearly isomorphous with thecrystal of the XAB5 Fv complex (Example 6), the structure of the latterwas used as input model for structure determination by molecularreplacement with the program Phaser. Iterative model correction andrefinement was performed with Coot (Crystallographic Object-OrientedToolkit) and Autobuster version 1.11.2 (Buster version 2.11.2), until nofurther significant improvements could be made to the crystallographicmodel. Final R- and R-free for all data were 0.197 and 0.253,respectively. The final refined model showed a root-mean-squaredeviation (RMSD) from ideal bond lengths and bond angles of 0.009 Å and1.0°, respectively.

(i) Results

The results of the X-ray refinement of the XAB4 Fv complex with humanIL-17A are provided in Table 16 and the three-dimensional structure ofthis complex is shown in FIG. 8. In this crystal structure, as in theXAB5 complex (Example 6), the XAB4 Fv complex has exact crystallographic2-fold symmetry: the asymmetric unit of the crystal contains only onehalf of the whole, dimeric complex. The XAB4 Fv makes contacts to bothIL-17A subunits, but the vast majority of the intermolecular contactsare to only one subunit (93% of the IL-17A surface buried by one XAB4 Fvis contributed by one subunit). The X-ray crystallography analysisconfirmed that the variant antibody XAB4 retained the target specificityand bound with high affinity to essentially the same epitope as theparental XAB1 antibody. However, in the XAB4 complex structure, as inthe XAB5 complex structure, the light-chain CDRL1 bears three pointmutations which provide enhanced binding to human IL-17A. As alreadydescribed for the XAB5 complex (Example 6), Trp 31 of the XAB4light-chain is engaged in strong hydrophobic/aromatic interactions withTyr 85 of IL-17A and, to a lesser extent, Phe 133 of IL-17A. Asn 30 ofthe XAB4 light-chain donates a H-bond to the main-chain carbonyl of Pro130 of IL-17A and is in van der Waals contact to Leu 49 (same IL-17Asubunit) and Val 45 (other IL-17A subunit). Glu 32 of the XAB4light-chain stabilizes the CDRL1 loop through intramolecular H-bondedinteractions. Furthermore, Glu 32 makes favorable electrostaticinteractions with Arg 124 of IL-17A, but is not engaged into a“head-to-head” salt-bridge interaction (FIG. 9). XAB4 also differs fromXAB1 in position 56 of the light-chain, as a result of an Asn to Glnmutation designed to remove a potential deamidation site. The X-rayanalysis shows that Gln 56 of XAB4 makes contacts to the protein antigenresidues Leu 76 and Trp 90, and reduces the solvent-accessibility of Tyr67 and Ser 64 (FIG. 10).

TABLE 16 X-ray refinement of the XAB4 Fv complex with IL-17A obtained bythe program Autobuster. REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM:BUSTER 2.11.2 REMARK 3 AUTHORS: BRICOGNE,BLANC, BRANDL, FLENSBURG,KELLER, REMARK 3     : PACIOREK, ROVERSI, SHARFF, SMART,     VONRHEIN,WOMACK; REMARK 3     : MATTHEWS, TEN EYCK, TRONRUD REMARK 3 REMARK 3DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS):3.15 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS): 78.15 REMARK 3 DATACUTOFF (SIGMA(F)): 0.0 REMARK 3 COMPLETENESS FOR RANGE (%): 99.85 REMARK3 NUMBER OF REFLECTIONS: 6881 REMARK 3 REMARK 3 FIT TO DATA USED INREFINEMENT. REMARK REMARK REMARK 3 CROSS-VALIDATION METHOD: THROUGHOUT 3FREE R VALUE TEST SET SELECTION: RANDOM 3 R VALUE (WORKING + TEST SET):0.1998 REMARK 3 R VALUE (WORKING SET): 0.1972 REMARK 3 FREE R VALUE:0.2531 REMARK 3 FREE R VALUE TEST SET SIZE (%): 5.01 REMARK 3 FREE RVALUE TEST SET COUNT: 345 REMARK 3 ESTIMATED ERROR OF FREE R VALUE: NULLREMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN. REMARK 3 TOTALNUMBER OF BINS USED: 5 REMARK 3 BIN RESOLUTION RANGE HIGH (ANGSTROMS):3.15 REMARK 3 BIN RESOLUTION RANGE LOW (ANGSTROMS): 3.52 REMARK 3 BINCOMPLETENESS (WORKING + TEST) (%): 99.85 REMARK 3 REFLECTIONS IN BIN(WORKING + TEST SET): 1916 REMARK 3 BIN R VALUE (WORKING + TEST SET):0.2376 REMARK 3 REFLECTIONS IN BIN (WORKING SET): 1820 REMARK 3 BIN RVALUE (WORKING SET): 0.2326 REMARK 3 BIN FREE R VALUE: 0.3295 REMARK 3BIN FREE R VALUE TEST SET SIZE (%): 5.01 REMARK 3 BIN FREE R VALUE TESTSET COUNT: 96 REMARK 3 ESTIMATED ERROR OF BIN FREE R VALUE: NULL REMARK3 REMARK 3 NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT. REMARK 3PROTEIN ATOMS: 2499 REMARK 3 NUCLEIC ACID ATOMS: 0 REMARK 3 HETEROGENATOMS: 5 REMARK 3 SOLVENT ATOMS: 0 REMARK 3 REMARK 3 B VALUES. REMARK 3FROM WILSON PLOT (A**2): 102.42 REMARK 3 MEAN B VALUE (OVERALL, A**2):124.95 REMARK 3 OVERALL ANISOTROPIC B VALUE. REMARK 3 B11 (A**2):−11.5511 REMARK 3 B22 (A**2): −28.0012 REMARK 3 B33 (A**2): 39.5523REMARK 3 B12 (A**2): 0.0000 REMARK 3 B13 (A**2): 0.0000 REMARK 3 B23(A**2): 0.0000 REMARK 3 REMARK 3 ESTIMATED COORDINATE ERROR. REMARK 3ESD FROM LUZZATI PLOT (A): 0.787 REMARK 3 DPI (BLOW EQ-9) BASED ON FREER VALUE (A): 0.474 REMARK 3 REMARK 3 REFERENCES: BLOW, D. (2002) ACTACRYST D58, 792-797 REMARK 3 REMARK 3 CORRELATION COEFFICIENTS. REMARK 3CORRELATION COEFFICIENT FO-FC: 0.9113 REMARK 3 CORRELATION COEFFICIENTFO-FC FREE: 0.8848 REMARK 3 REMARK 3 X-RAY WEIGHT: 20.89 REMARK 3 REMARK3 GEOMETRY FUNCTION. REMARK 3 RESTRAINT LIBRARIES. REMARK 3 NUMBER OFLIBRARIES USED: 8 REMARK 3 LIBRARY 1: protgeo_eh99.dat (V1.8) 20110121STANDARD REMARK 3     AMINO ACID DICTIONARY. BONDS     AND ANGLES FROMREMARK 3     ENGH AND HUBER EH99. OTHER     VALUES BASED ON REMARK 3    PREVIOUS TNT OR TAKEN     FROM CCP4. INCLUDES REMARK 3     HYDROGENATOMS. REMARK 3 LIBRARY 2: exoticaa.dat (V1.8) 20100430 COLLECTION OFREMARK 3     NON-STANDARD AMINO ACIDS,     MAINLY EH91 WITHOUT REMARK 3    IDEAL DISTANCE INFO REMARK 3 LIBRARY 3: nuclgeo.dat (V1.14) 20091104REMARK 3 LIBRARY 4: bcorrel.dat (V1.15) 20080423 REMARK 3 LIBRARY 5:contact.dat (V1.20.2.1) 20110510 REMARK 3 LIBRARY 6:idealdist_contact.dat (V1.7) 20110119 REMARK 3     IDEAL-DISTANCECONTACT     TERM DATA AS USED IN REMARK 3     PROLSQ. VALUES USED HEREARE     BASED ON THE REFMAC REMARK 3     5.5 IMPLEMENTATION. REMARK 3LIBRARY 7: restraints for SO4 (SULFATE ION) from cif REMARK 3    dictionary 504.cif using refmacdict2tnt REMARK 3     revision1.23.2.7; buster common-compounds REMARK 3     v 1.0 (5 May 2011) REMARK3 LIBRARY 8: assume.dat (V1.10) 20110113 REMARK 3 REMARK 3 NUMBER OFGEOMETRIC FUNCTION TERMS DEFINED: 15 REMARK 3 TERM COUNT WEIGHTFUNCTION. REMARK 3 BOND LENGTHS: 2566; 2.00; HARMONIC REMARK 3 BONDANGLES: 3486; 2.00; HARMONIC REMARK 3 TORSION ANGLES: 860; 2.00;SINUSOIDAL REMARK 3 TRIGONAL CARBON PLANES: 61; 2.00; HARMONIC REMARK 3GENERAL PLANES: 369; 5.00; HARMONIC REMARK 3 ISOTROPIC THERMAL FACTORS:2566; 20.00; HARMONIC REMARK 3 BAD NON-BONDED CONTACTS: NULL; NULL; NULLREMARK 3 IMPROPER TORSIONS: NULL; NULL; NULL REMARK 3 PSEUDOROTATIONANGLES: NULL; NULL; NULL REMARK 3 CHIRAL IMPROPER TORSION: 323; 5.00;SEMIHARMONIC REMARK 3 SUM OF OCCUPANCIES: NULL; NULL; NULL REMARK 3UTILITY DISTANCES: NULL; NULL; NULL REMARK 3 UTILITY ANGLES: NULL; NULL;NULL REMARK 3 UTILITY TORSION: NULL; NULL; NULL REMARK 3 IDEAL-DISTCONTACT TERM: 2984; 4.00; SEMIHARMONIC REMARK 3 REMARK 3 RMS DEVIATIONSFROM IDEAL VALUES. REMARK 3 BOND LENGTHS (A): 0.009 REMARK 3 BOND ANGLES(DEGREES): 1.00 REMARK 3 PEPTIDE OMEGA TORSION ANGLES (DEGREES): 4.39REMARK 3 OTHER TORSION ANGLES (DEGREES): 18.96 REMARK 3 REMARK 3SIMILARITY. REMARK 3 NCS. REMARK 3 NCS REPRESENTATION: NONE REMARK 3TARGET RESTRAINTS. REMARK 3 TARGET REPRESENTATION: LSSR REMARK 3 TARGETSTRUCTURE: xab5_il17a_complex_final_buster.pdb REMARK 3 REMARK 3 TLSDETAILS. REMARK 3 NUMBER OF TLS GROUPS: 3 REMARK 3 REMARK 3 TLS GROUP: 1REMARK 3 SET: { H|* } REMARK 3 ORIGIN FOR THE GROUP (A): 10.9676 −8.7396−10.1379 REMARK 3 T TENSOR REMARK 3 T11: −0.1266 T22: 0.0257 REMARK 3T33: −0.2829 T12: −0.3040 REMARK 3 T13: −0.0312 T23: 0.1050 REMARK 3 LTENSOR REMARK 3 L11: 7.4496 L22: 4.4770 REMARK 3 L33: 4.2880 L12: 1.1123REMARK 3 L13: −1.8044 L23: 3.0307 REMARK 3 S TENSOR REMARK 3 S11: 0.2013S12: 0.3070 S13: −0.5774 REMARK 3 S21: 0.4752 S22: −0.5377 S23: 0.7096REMARK 3 S31: 1.0885 S32: −1.0885 S33: 0.3364 REMARK 3 REMARK 3 TLSGROUP: 2 REMARK 3 SET: { I|* } REMARK 3 ORIGIN FOR THE GROUP (A):22.7365 0.7101 −35.1243 REMARK 3 T TENSOR REMARK 3 T11: −0.1883 T22:0.1529 REMARK 3 T33: −0.3560 T12: 0.0318 REMARK 3 T13: −0.1985 T23:0.0144 REMARK 3 L TENSOR REMARK 3 L11: 2.7494 L22: 9.3427 REMARK 3 L33:3.8648 L12: 0.8073 REMARK 3 L13: −0.6650 L23: −2.0544 REMARK 3 S TENSORREMARK 3 S11: 0.0485 S12: 0.3188 S13: 0.0579 REMARK 3 S21: 0.0595 S22:0.1433 S23: 0.7000 REMARK 3 S31: 0.0050 S32: −0.6066 S33: −0.1917 REMARK3 REMARK 3 TLS GROUP: 3 REMARK 3 SET: { L|* } REMARK 3 ORIGIN FOR THEGROUP (A): 33.2517 −11.1794 −14.2151 REMARK 3 T TENSOR REMARK 3 T11:0.0667T 22: −0.1645 REMARK 3 T33: −0.2360 T12: 0.1870 REMARK 3 T13:−0.2270 T23: −0.1209 REMARK 3 L TENSOR REMARK 3 L11: 3.3694 L22: 3.7848REMARK 3 L33: 8.8916 L12: −0.6497 REMARK 3 L13: −2.6132 L23: 0.8234REMARK 3 S TENSOR REMARK 3 S11: −0.0839 S12: −0.2629 S13: −0.1560 REMARK3 S21: 0.3804 S22: 0.7574 S23: −0.5378 REMARK 3 S31: 1.0885 S32: 1.0885S33: −0.6736 REMARK 3 REMARK 3 REFINEMENT NOTES. REMARK 3 NUMBER OFREFINEMENT NOTES: 1 REMARK 3 NOTE 1: IDEAL-DIST CONTACT TERM CONTACTSETUP. ALL ATOMS REMARK 3   HAVE CCP4 ATOM TYPE FROM LIBRARY REMARK 3REMARK 3 OTHER REFINEMENT REMARKS: NULL REMARK 3 SSBOND 1 CYS H 22 CYS H96 1555 1555 2.03 SSBOND 2 CYS I 94 CYS I 144 1555 1555 2.05 SSBOND 3CYS I 99 CYS I 146 1555 1555 2.04 SSBOND 4 CYS L 23 CYS L 88 1555 15552.07 CISPEP 1 TYR I 85 PRO I 86 0 3.67 CISPEP 2 GLU I 125 PRO I 126 0−9.25 CISPEP 3 PRO I 126 PRO I 127 0 5.92 CISPEP 4 SER L 7 PRO L 8 0−6.50 CISPEP 5 TYR L 94 PRO L 95 0 −6.52 CRYST1 55.760 87.109 156.30690.00 90.00 90.00 C 2 2 21

FIG. 8 provides the three-dimensional structure of the XAB4 Fv complexwith human IL-17A. FIG. 8A shows the two XAB4 Fv fragments inspace-filling representation, and the IL-17A homodimer is shown incartoon representation. FIG. 8B shows the two XAB4 Fv fragments incartoon representation, and the IL-17A homodimer is shown inspace-filling representation. The heavy- and light-chain of the XAB4 Fvare shown in dark and light grey, respectively. One chain of the IL-17Ahomodimer is shown in light grey, the other is shown in dark grey.

FIG. 9 provides the three-dimensional structure of the XAB4 Fv complexwith human IL-17A as a close-up view of the antibody L-CDR1 bearing thethree mutations found by the structure-guided biased library approach:Asn 30, Trp 31 and Glu 32. These XAB4 side-chains contribute new bindinginteractions to the antigen human IL-17A, in particular to IL-17Aresidues Tyr85, Phe133, Arg124, Pro 130, Leu 49 (all from the sameIL-17A subunit) and Val 45 (from the other IL-17A subunit).

FIG. 10 provides the three-dimensional structure of the XAB4 Fv complexwith human IL-17A as a close-up view of the antibody L-CDR2 showing theAsn 56 to Gln mutation. This XAB4 side-chain contributes bindingcontacts to IL-17A residues Trp 90 and Leu 76, and reduces thesolvent-accessibility of Tyr 67 and Ser 64 (all from the same IL-17Asubunit).

To summarize, X-ray crystallography analysis confirmed that the variantantibodies selected for further analysis retained their targetspecificity and bound with high affinity to essentially the same epitopeas the parental XAB1 antibody. Tighter binding between each of thevariant antibodies and IL-17A was observed, as a result of additional orimproved binding contacts (see Table 17 below).

Further characterisation of the variant antibodies was conducted asdescribed below.

TABLE 17 X-ray analyses of the IL-17A epitope bound by XAB1, XAB2, XAB4and XAB5: summary and structure-based, qualitative classification ofepitope residues. (*): residue contributed by the second IL-17A subunitEpitope residue class XAB1 XAB2 XAB4 XAB5 Very important Arg 78, Glu Arg78, Glu Arg 78, Glu Arg 78, Glu epitope 80, Trp 90 80, Trp 90 80, Tyr85, Trp 80, Tyr 85, Trp residues 90, Arg 124 90, Arg 124 Other Pro 82,Ser Arg 43*, Pro Pro 82, Ser Pro 82, Ser important 87, Val 88, 82, Ser87, 87, Val 88 87, Val 88 epitope Arg 124 Val 88, Arg residues 124Additional Val 45*, Leu Pro 42*, Val Val 45*, Leu Val 45*, Leucontributions 49, Ile 51, Asp 45*, Leu 49, 49, Asp 81, 49, Asp 81, 81,Glu 83, Ile 51, Asp 81, Glu 83, Pro Glu 83, Pro Tyr 85, Asn Glu 83, Tyr86, Pro 130, 86, Pro 130, 131, Lys 137* 85, Asn 131, Phe 133, Lys Phe133, Lys Lys 137* 137* 137* Little or no Thr 44*, Leu Leu 76, His Arg43*, Asn Arg 43*, Asn direct 76, His 77, 77, Asn 79, 50, Ser 64, 50, Leu76, contribution Asn 79, Arg Arg 84, Pro Tyr 67, Leu His 77, Asn 84, Pro86, 86, Lys 93, 76, His 77, 79, Arg 84, Lys 93, Glu Glu 118*, Pro Asn79, Arg Glu 118*, Leu 118*, Pro 130, 130, Phe 133 84, Glu 118*, 122, Asn131, Phe 133 Leu 122, Asn Leu 135* 131, Leu 135*

Example 8. Affinity Measurements and Cross-Reactivity Measured byBiacore™

Determination of kinetic binding parameters was achieved by surfaceplasmon resonance measurements using the optical biosensor Biacore™ T200or T100 (http://www.biacore.com). This technology allows the label-freedetermination of the microscopic rate constants for binding (k_(a)) anddissociation (k_(d)) of a ligand to a receptor. It is thereforeespecially suited for characterizing the antibody-antigen interactions.

Indirect binding of antibodies to the Biacore™ chip surface was done viaan anti-human Ig antibody (GE Healthcare Bio-Sciences AB; Cat. No.BR-1008-39) 25 μg/ml in immobilization buffer (10 mM Sodium acetate pH5.0) or through protein A (RepliGen: rPA-50) 20 μg/ml in immobilizationbuffer (10 mM Sodium acetate pH 5.0 or pH 4.0).

Antibody was diluted into blank buffer to a final concentration of 1.00or 1.25 μg/ml.

Affinity measurements for the determination of dissociation constants ofXAB4 or XAB1 was performed for recombinant huIL-17A (SEQ ID NO: 78, e.g.2-fold increasing concentrations from 0.14 to 8.8 nM), recombinanthuIL-17A/F heterodimer (e.g. 2-fold increasing concentrations from 0.13to 8 nM), recombinant huIL-17F (SEQ ID NO: 77; e.g. 2-fold increasingconcentrations from 7.8 to 500 nM) cynomolgus IL-17A (SEQ ID NO: 79;e.g. 2-fold increasing concentrations from 0.63 to 40 nM) rhesus IL-17A(SEQ ID NO: 82; e.g. 2-fold increasing concentrations from 1.6 to 100nM), marmoset IL-17A (SEQ ID NO: 82; e.g. 2-fold increasingconcentrations from 0.63 to 40 nM), recombinant mIL-17A (SEQ ID NO: 83;e.g. 2-fold increasing concentrations from 0.78 to 50 nM), recombinantmIL-17A/F (R&D Systems® Cat #5390-IL; e.g. 2-fold increasingconcentrations from 1.25 to 40 nM) rat IL-17A (SEQ ID NO: 85; e.g.2-fold increasing concentrations from 0.78 to 50 nM), using the indirectcoupling/binding method (see above) and surface was regenerated with 10mM glycine pH 1.75 or MgCl₂ (3 M). One chip surface was coated andreused without significant loss of binding capacity. Ligandconcentrations were chosen to start below the K_(D) and to end at aconcentration higher than ten times the K_(D).

Similar but not identical conditions were used to measure affinity ofXAB2 and XAB3.

The kinetic traces were evaluated with the Biacore™ T200 ControlSoftware version 1.0. The full set of these traces with increasingconcentrations is taken together and is called a run. Two zeroconcentration samples (blank runs) were included in each analyteconcentration series to allow double-referencing during data evaluation

Results

The binding of the anti-IL-17 antibodies XAB4, XAB1, XAB2 and XAB3 tohuman, cynomolgus monkey, marmoset monkey, rhesus monkey, mouse and ratIL-17A, to human and mouse IL-17A/F heterodimer and to human IL-17F wasdetermined by surface plasmon resonance using the Biacore™ technology.

The kinetic rate constants for association (k_(a)) and dissociation(k_(d)), as well as the dissociation equilibrium constant (K_(D)) werecalculated.

The affinity data of XAB4 is shown in Table 18, the affinity data ofXAB1 is shown in

Table 19, the affinity data of XAB2 is shown in Table 20, and theaffinity data of XAB3 is shown in Table 21. Affinity maturation of XAB1,XAB2 and XAB3 increased the affinity towards human, cynomolgus monkey,mouse and rat IL-17A.

TABLE 18 Affinity and kinetic rate constants of XAB4 binding. Antigenk_(a) (1/MS) k_(d) (1/S) K_(D) (M) hulL-17A 4.1 ± 0.1 E+06 2.3 ± 0.1E−05 5.7 ± 0.0 E−12 hulL-17A/F 8.9 ± 0.2 E+5  <1.0 ± 0.0 E−05* <1.1 ±0.0 E−11* hulL-17F n.d. n.d. n.d. cynolL-17A 4.1 ± 0.5 E+05 1.3 ± 0.0E−05 3.1 ± 0.4 E−11 marmIL-17A 1.2 ± 0.0 E+06 2.2 ± 0.0 E−05 1.8 ± 0.0E−11 rhesIL-17A 3.0 ± 0.1 E+05 1.2 ± 0.1 E−05 4.0 ± 0.1 E−11 mIL-17A 3.8± 0.1 E+05 6.2 ± 0.3 E−05 1.6 ± 0.1 E−10 mIL-17A/F 2.421E+05 6.305E−052.604 E−10 ratIL-17A 5.5 ± 0.4 E+05 4.6 ± 0.9 E−05 8.4 ± 1.0 E−11 n.d. =not determinable, applied antigen conc. range too low and non-specificbinding of antigen to reference flow cell observed at the highestantigen concentrations (500-50 pM). *dissociation rate outside thelimits that can be measured by the instrument (k_(d) <1 × 10⁻⁵ 1/s)

TABLE 19 Affinity and kinetic rate constants of XAB1 binding. Antigenk_(a) (1/MS) k_(d) (1/s) K_(D) (M) hul L-17A   2.33E+06  9.39E−05 4.03E−11 hul L-17A/F  9.097E+05  0.001342 1.475E−09 hulL-17F n.d. n.d.n.d. cynolL-17A   2.14E+05  1.13E−04  5.26E−10 rhesIL-17A   8.87E+05 9.97E−05  1.12E−09 mIL-17A   4.05E+05  1.43E−04  3.53E−10 mIL-17A/F1.8757E+05 9.547E−04 5.093E−09 ratIL-17A  5.44E+05  1.64E−04  3.01E−10n.d. = not determinable, applied antigen conc. range too low andnon-specific binding of (500-50 antigen to reference flow cell observedat three highest antigen concentrations pM).

TABLE 20 Affinity and kinetic rate constants of XAB2 binding. Antigenk_(a) (1/MS) k_(d) (1/S) K_(D) (M) hulL-17A 4.09E+06 7.12E−05 1.76E−11

TABLE 21 Affinity and kinetic rate constants of XAB3 binding. Antigenk_(a) (1/Ms) k_(d) (1/s) K_(D) (M) huIL-17A 5.48E+06 5.01E−05 9.58E−12huIL-17A/F 3.37E+06 1.03E−04 3.29E−11 huIL-17F n.d. n.d. n.d. cynoIL-17A1.21E+06 4.23E−05 3.49E−11 mIL-17A 5.87E+05 1.01E−04 1.74E−10 ratIL-17A9.05E+05 7.59E−05 8.26E−11 n.d. = not determinable

The affinities and kinetic rate constants for XAB2, XAB3 and XAB5 arecomparable to those observed for XAB4.

Example 9. Binding in ELISA to IL-17A and Other Family Members

A titration of the antibodies of interest on different antigens wascarried out. Briefly, wells of ELISA microtiter plates (Nunc Immunoplates MaxiSorp: Invitrogen, Cat #4-39454 Å) were coated with 1 μg/ml ofrecombinant huIL-17A (SEQ ID NO: 76; 1.8 mg/ml), recombinant huIL-17A/F(0.59 mg/ml), recombinant huIL-17F (SEQ ID NO: 77; 1.8 mg/ml)),recombinant huIL-17B (R&D Systems® Cat #12481B/CF), recombinant huIL-17C(R&D Systems® Cat #12341L/CF), recombinant huIL-17D (R&D Systems® Cat#15041L/CF), recombinant huIL-17E (R&D Systems® Cat #1258-IL/CF),recombinant cynoIL-17A (SEQ ID NO: 79; 0.21 mg/ml), recombinantcynoIL-17F (SEQ ID NO: 80; 1.525 mg/ml), recombinant mIL-17A (SEQ ID NO:83; 2.8 mg/ml), recombinant mIL-17A/F (R&D Systems® Cat #5390-IL),recombinant mIL-17F (SEQ ID NO: 84; 0.2 mg/ml) and recombinant ratIL-17A(SEQ ID NO: 85; 3.8 mg/ml) (100 μl/well) in phosphate buffered saline(PBS) without Ca and Mg (10×, Invitrogen Cat #14200-083) 0.02% NaN₃(Sigma Cat # S-8032) and incubated overnight at 4° C.

The following day, microtiter plates were blocked with 300 μl of PBS/2%BSA (fraction V; Roche Cat #10 735 094 001)/0.02% NaN₃ for 1 h at 37° C.Plates were then washed 4 times with PBS/0.05% Tween 20 (Sigma Cat #P7949)/0.02% NaN₃. XAB4 or XAB1 were added at 1 μg/ml in triplicatewells (100 μl/well) for 3 h at room temperature.

To verify coating of antigens to the plates, control antibodies wereused and in particular, a mouse mAb anti-huIL-17F, (Novartis, 5 μg/ml) agoat anti-hu-IL-17B (R&D Systems® Cat # AF1248; 10 μg/ml), a mouse mAbanti-huIL-170 (R&D Systems® Cat # MAB1234; 10 μg/ml), a goatanti-huIL-17D (R&D Systems® Cat # AF1504; 10 μg/ml), a mouse mAb antihu-IL-17E (R&D Systems® Cat # MAB1258, 10 μg/ml), a mouse anti-mIL-17Aor anti-mIL-17A/F (Novartis; 1 μg/ml), and a rat anti-mIL-17F (R&DSystems® Cat # MAB2057; 1 μg/ml;) (100 μl/well in PBS, 0.02% NaN₃ for 3hat RT).

Plates were then washed 4 times with PBS/0.05% Tween 20/0.02% NaN₃.Then, an alkaline phosphatase-conjugated goat anti-human IgG antibody(Sigma Cat # A9544) was added to the wells that received test antibodyat a dilution of 1/20000 (100 μl/well) for 2h 30 min at RT. To thewells, that received mouse mAb, an alkaline phosphatase-conjugated goatanti-mouse IgG antibody (Sigma Cat # A7434) was added at a dilution of1/10000 (100 μl/well) for 2 h 30 min at RT. An alkaline phosphataseconjugated mouse anti goat IgG antibody (Sigma Cat # A8062) was added tothe goat antibodies at a dilution of 1/50000 (100 μl/well) for 2 h 30min at RT. Plates were then washed 4 times and 100 μl of the substrate(p-nitrophenyl phosphate tablets; Sigma; 5 mg Cat # N9389; 20 mg Cat #.N2765) dissolved in diethanolamine buffer pH 9.8, to give a finalconcentration of 1 mg/ml, were added to each well.

Plates were read after 30 min in a Spectra Max M5 Microplate Reader(Molecular Devices) using filters of 405 and 490 nm. Values are themeans±SEM of triplicate values.

Results

These studies show that XAB4 and XAB1 are able to bind human and mouseIL-17A, and human and mouse IL-17A/F. In addition it is shown that XAB4is able to bind cynomolgus and rat IL-17A. Binding to human, cynomolgusand mouse IL-17F was not detected under these experimental conditions aswell as binding to other human family members (IL-17B, IL-17C, IL-17Dand IL-17E).

TABLE 22 Cross-reactivity of XAB4 and XAB1 to human, cynomolgus monkey,mouse and rat IL-17 family members, by ELISA. XAB4 Control antibody XAB1Control antibody (1 μg/ml) (1 or 10 μg/ml) (1 μg/ml) (1 or 10 μg/ml) O.Dvalues O.D values O.D values O.D values (mean ± SEM) (mean ± SEM) (mean± SEM) (mean ± SEM) hu IL-17A 2.471 ± 0.0448 1.302 ± 0.0554 hu IL-17A/F2.137 ± 0.0429 1.222 ± 0.0202 hu IL-17F 0.049 ± 0.0056 0.032 ± 0.00051.913 ± 0.0483 hu IL-17B 0.034 ± 0.0007 0.283 ± 0.0066 0.049 ± 0.00131.441 ± 0.0283 hu IL-17C 0.036 ± 0.0002 0.290 ± 0.0027 0.032 ± 0.00020.558 ± 0.0169 hu IL-17D 0.034 ± 0.0005 0.292 ± 0.0048 0.031 ± 0.00100.867 ± 0.0372 hu IL-17E 0.035 ± 0.0014 0.833 ± 0.0239 0.033 ± 0.00032.054 ± 0.0378 cyno IL-17A 1.926 ± 0.0355 cyno IL-17F 0.085 ± 0.0336mouse IL-17A 1.585 ± 0.0428 1.086 ± 0.0119 1.439 ± 0.0354 3.697 ± 0.0602mouse IL-17A/F 2.263 ± 0.0243 1.142 ± 0.0315 1.762 ± 0.0097 2.084 ±0.0223 mouse IL-17F 0.098 ± 0.0060 1.294 ± 0.0134 0.044 ± 0.0008 1.770 ±0.0302 rat IL-17A 1.772 ± 0.1668

Example 10. Cross-Reactivity to Other Human, Mouse and Rat Interleukinsby ELISA

In another set of experiments the cross-reactivity of antibodies of thedisclosure for selected human, mouse or rat cytokines was evaluated.

Triplicate wells of ELISA microtiter plates (Nunc Immuno platesMaxiSorp: Invitrogen Cat #4-39454A) were coated with 100 μl/well of thefollowing cytokines: recombinant huIL1β (Novartis), recombinant huIL-3(R&D Systems® Cat #203-IL/CF), recombinant huIL-4 (R&D Systems® Cat#204-IL/CF), recombinant huIL-6 (R&D Systems® Cat #206-IL-1010/CF),recombinant huIL-8 (R&D Systems® Cat #208-IL-010/CF), recombinanthuIL-12 (R&D Systems® Cat #219-IL-005/CF), recombinant huIL-13(Novartis), recombinant huIL-17A (SEQ ID NO: 76), recombinanthuIL-17A/F, recombinant huIL-17F (SEQ ID NO: 77), recombinant huIL-18(MBL Cat # B003-5), recombinant huIL-20 (Novartis), recombinant huIL-23(R&D Systems® Cat #1290-IL-010/CF), recombinant huIFNγ (Roche),recombinant huTNFα (Novartis), recombinant huEGF (Sigma Cat # E9644.),recombinant huTGF62 (Novartis), recombinant mIL-1β (R&D Systems® Cat#401-ML), recombinant mIL-2 (R&D Systems® 402-ML-020/CF), recombinantmIL-6 (R&D Systems® Cat #406-ML-010/CF), recombinant mIL-12 (R&DSystems® Cat #419-ML-010/CF), recombinant mIL-17A (SEQ ID NO: 83),recombinant mIL-17A/F (R&D Systems® Cat #5390-IL), recombinant mIL-17F(R&D Systems® Cat #2057—IL/CF), recombinant mIL-18 (MBL Cat # B004-5),recombinant mIL-23 (R&D Systems® Cat #1887-ML), recombinant mIFN-γ (R&DSystems® Cat #485-MT), recombinant mTNFα (R&D Systems® Cat #410-MT),recombinant rat IL-4 (R&D Systems® Cat #504-RL/CF), recombinant rat IL-6(R&D Systems® Cat #506-RL-010), recombinant ratIL-12 (R&D Systems® Cat#1760-RL/CF), recombinant ratIL-17A (SEQ ID NO: 85), recombinantratIL-23 (R&D Systems® Cat #3136-RL-010/CF), recombinant ratTNFα (R&DSystems® Cat #510-RT/CF), at 1 μg/ml with the exception of recombinantmIL-6, recombinant mIL-12 and recombinant mTNFα which were coated at 0.5μg/ml in phosphate buffered saline (PBS) without Ca and Mg (10×,Invitrogen Cat #14200-083) 0.02% NaN₃ (Sigma Cat # S-8032) and incubatedovernight at 4° C.

The following day, microtiter plates were blocked with 300 μl of PBS/2%BSA (fraction V; Roche Cat #10 735 094 001)/0.02% NaN₃ for 1 h at 37° C.Plates were then washed 4 times with PBS/0.05% Tween 20 (Sigma Cat #P7949)/0.02% NaN₃.

The antibodies of the disclosure were added at 10 μg/ml (100 μl/well)for 3 h at room temperature. To verify coating of antigens to theplates, 100 μl/well of the following control antibodies were used: amouse anti-huIL1β (R&D Systems® Cat # MAB601), a mouse anti-huIL-3 (R&DSystems® Cat # MAB603), a mouse anti-huIL4 (R&D Systems® Cat # MAB604),a mouse anti-huIL-6 (R&D Systems® Cat # MAB206), a mouse anti-hu-IL8(R&D Systems® Cat # MAB208), a mouse anti-huIL-12 (R&D Systems® Cat #MAB219), a mouse anti-huIL-13 (Novartis), a mouse anti-huIL-17A(Novartis), a mouse anti-huIL-17F (Novartis), a mouse anti-huIL-18 (MBLCat # D043-3), a mouse anti-huIL-20 (Abcam Cat # ab57227), a goatanti-huIL-23 (R&D Systems® Cat # AF1716), a mouse anti-huIFN-γ (R&DSystems® Cat # MAB285), a mouse anti-huTNF-α (R&D Systems® Cat #MAB610), a mouse anti-hu-EGF (R&D Systems® Cat # MAB236), a humananti-huTGF62 (Novartis), a rat anti-mIL-1β (R&D Systems® Cat # MAB401),a rat anti-mIL-2 (R&D Systems® Cat # MAB402), a rat anti-mIL-6 (R&DSystems® Cat # MAB406), a rat anti-mIL-12 (R&D Systems® Cat # MAB419), amouse anti-m/ratIL-17A (Novartis), a rat anti-mIL-17F (R&D Systems® Cat# MAB2057), a rat anti-mIL-18 (MBL Cat # D047-3), a rat anti-mIFN-γ (R&DSystems® Cat # MAB485), a goat anti-mTNFα (R&D Systems® Cat #AF-410-NA), a mouse anti-rat IL-4 (R&D Systems® Cat # MAB504), a goatanti-rat IL-6 (R&D Systems® Cat # AF506), a goat anti-rat IL-12 (R&DSystems® Cat # AF1760), a mouse anti-rat IL-23 (R&D Systems® Cat #MAB3510), a mouse anti-rat TNFα (R&D Systems® Cat # MAB510). They wereadded at 1 or 5 μg/ml, in PBS, 0.02% NaN₃ for 3 h at RT.

Plates were then washed 4 times with PBS/0.05% Tween 20/0.02% NaN₃.Then, an alkaline phosphatase-conjugated goat anti-human IgG antibody(Sigma Cat # A9544) was added to the wells with human antibodies at adilution of 1/20000 (100 μl/well). An alkaline phosphatase-conjugatedgoat anti-mouse IgG antibody (Sigma Cat # A1047) was added to the wellswith mouse antibodies at a dilution of 1/10000 (100 μl/well). Analkaline phosphatase-conjugated rabbit anti-goat IgG antibody (Sigma Cat# A7650) was added to the wells with goat antibodies at a dilution of1/1000 (100 μl/well) and an alkaline phosphatase-conjugated rabbit antirat-IgG antibody (Sigma Cat # A6066) was added to the wells with ratantibodies at a dilution of 1/20000 (100 μl/well). The secondaryantibodies were incubated for 2 h 30 min at RT. Plates were then washed4 times and 100 μl of the substrate (p-nitrophenyl phosphate tablets;Sigma; 5 mg Cat #. N9389 or 20 mg Cat # N2765) dissolved indiethanolamine buffer pH 9.8, to give a final concentration of 1 mg/ml,were added to each well.

Plates were read after 30 min at RT or ON at 4° C. in a Spectra Max M5Microplate Reader (Molecular Devices) using filters of 405 and 490 nm.Values are the means±SEM of triplicate values.

Results

The data obtained show that both XAB4 and XAB1 are highly selective forIL-17A of human, mouse and rat origin and for IL-17A/F of human andmouse origin. In addition, under the conditions tested, the reactivityof XAB1 at 10 μg/ml for human IL-17F (not seen at 1 μg/ml, see above) isnot observed with XAB4. Reactivity for the other cytokines tested wasnot detected.

TABLE 23 Cross-reactivity of XAB4 and XAB1 to human cytokines by ELISA.XAB4 Control antibody XAB1 Control antibody (10 μg/ml) (5 μg/ml) (10μg/ml) (1 μg/ml) O.D values O.D values O.D values O.D values (mean ±SEM) (mean ± SEM) (mean ± SEM) (mean ± SEM) IL1β 0.015 ± 0.0075 0.867 ±0.0107 −0.110 ± 0.0901  3.071 ± 0.0486 IL3 0.167 ± 0.1288 0.732 ± 0.0194−0.049 ± 0.0738  2.931 ± 0.0779 IL4 0.047 ± 0.0089 0.806 ± 0.0617 0.057± 0.0458 2.555 ± 0.1499 IL6 −0.015 ± 0.0103  1.452 ± 0.2020 −0.044 ±0.0838  2.976 ± 0.1025 IL8 0.018 ± 0.0078 3.130 ± 0.0109 0.058 ± 0.04313.153 ± 0.1228 IL12 0.009 ± 0.0058 0.853 ± 0.0496 −0.097 ± 0.1600  2.964± 0.1370 IL13 0.019 ± 0.0085 2.639 ± 0.0309 0.125 ± 0.0706 2.639 ±0.0309 IL17A 3.178 ± 0.0697 3.136 ± 0.0644 2.745 ± 0.0879 2.731 ± 0.0850IL17A/F 3.100 ± 0.0458 3.024 ± 0.0816 2.644 ± 0.2517 3.024 ± 0.0816IL17F 0.035 ± 0.0138 3.114 ± 0.0672 0.613 ± 0.4162 3.185 ± 0.0110 IL18−0.001 ± 0.0234  3.313 ± 0.2080 −0.086 ± 0.0170  3.313 ± 0.2080 IL200.039 ± 0.0117 3.039 ± 0.0671 0.335 ± 0.2442 3.118 ± 0.0252 IL23 −0.022± 0.0450  3.435 ± 0.0878 0.085 ± 0.0678 3.350 ± 0.0886 IFN-γ 0.048 ±0.0676 3.419 ± 0.0404 0.059 ± 0.0511 3.236 ± 0.0312 TNF-α 0.009 ± 0.01973.373 ± 0.0550 0.289 ± 0.0318 3.275 ± 0.0440 EGF 0.126 ± 0.0858 3.432 ±0.1050 0.062 ± 0.0427 3.233 ± 0.1126 TGFβ2 0.018 ± 0.0190 3.397 ± 0.03580.146 ± 0.0653 3.246 ± 0.0303 BSA 0.009 ± 0.0194 0.010 ± 0.0192 0.043 ±0.0033 0.149 ± 0.0558 N.B. the negative values are due to the fact thatthe blank (O.D. value of wells without specific antibodies) issubtracted.

TABLE 24 Cross-reactivity of XAB4 and XAB1 to mouse cytokines by ELISA.XAB4 Control antibody XAB1 Control antibody (10 μg/ml) (5 μg/ml) (10μg/ml) (5 μg/ml) O.D values O.D values O.D values O.D values (mean ±SEM) (mean ± SEM) (mean ± SEM) (mean ± SEM) IL-1β 0.022 ± 0.0057 0.611 ±0.0665 0.007 ± 0.0123 0.624 ± 0.0455 IL2 0.024 ± 0.0227 3.548 ± 0.12830.022 ± 0.0125 3.295 ± 0.0557 IL6 0.031 ± 0.0063 3.291 ± 0.0174 0.038 ±0.0091 3.340 ± 0.1115 IL12 0.035 ± 0.0110 3.359 ± 0.0094 −0.005 ±0.0121  3.295 ± 0.0331 IL17A 3.285 ± 0.0445 3.180 ± 0.0702 2.974 ±0.0281 3.186 ± 0.0505 IL17A/F 3.342 ± 0.1047 3.407 ± 0.1102 3.169 ±0.0340 3.214 ± 0.0145 IL17F 0.034 ± 0.0122 3.359 ± 0.0247 −0.058 ±0.0326  3.264 ± 0.0309 IL18 0.054 ± 0.0149 2.650 ± 0.0227 0.022 ± 0.01232.572 ± 0.0145 IL23 0.058 ± 0.0139 0.601 ± 0.0314 0.009 ± 0.0007 0.590 ±0.0378 IFN-γ 0.038 ± 0.0114 2.751 ± 0.0515 0.048 ± 0.0063 2.388 ± 0.2351TNF-α 0.065 ± 0.0154 3.258 ± 0.1097 0.025 ± 0.0081 3.476 ± 0.0714 BSA0.015 ± 0.0078 0.035 ± 0.0047 0.015 ± 0.0078 0.035 ± 0.0047 N.B. thenegative values are due to the fact that the blank (O.D. value of wellswithout specific antibodies) is subtracted.

TABLE 25 Cross-reactivity of XAB4 and XAB1 to rat cytokines by ELISA.XAB4 Control antibody XAB1 Control antibody (10 μg/ml) (5 μg/ml) (10μg/ml) (5 μg/ml) O.D values O.D values O.D values O.D values (mean ±SEM) (mean ± SEM) (mean ± SEM) (mean ± SEM) IL4 0.026 ± 0.0082 3.168 ±0.0297 0.017 ± 0.0092 3.324 ± 0.1092 IL6 0.021 ± 0.0028 3.116 ± 0.03180.000 ± 0.0141 3.253 ± 0.1078 IL12 0.009 ± 0.0113 3.185 ± 0.0921 −0.007± 0.0082  3.310 ± 0.0692 IL17A 3.483 ± 0.0910 3.156 ± 0.0890 1.202 ±0.0136 3.359 ± 0.0670 IL23 0.023 ± 0.0050 3.380 ± 0.2127 0.011 ± 0.00103.199 ± 0.1078 TNF-α 0.020 ± 0.0104 3.346 ± 0.1376 0.003 ± 0.0029 3.159± 0.0854 BSA 0.015 ± 0.0078 0.035 ± 0.0047 0.015 ± 0.0078 0.035 ± 0.0047N.B. the negative values are due to the fact that the blank (O.D. valueof wells without specific antibodies) is subtracted.

Example 11. IL-17A—IL-17RA and IL-17A/F-IL-17RA In Vitro CompetitiveBinding Inhibition Assay

Human IL-17RA was used from a stock solution (BTP22599: 1.68 mg/ml=46.2μM). ELISA microtiter plates were coated with human IL-17RA (100μl/well, 1 μg/ml, ˜27.5 nM) in PBS/0.02% NaN₃ and incubated overnight atroom temperature. The following day the plates were blocked with 300 μlof PBS/2% BSA/0.02% NaN₃ for 1 h at 37° C. Then the plates were washed 4times with PBS/0.05% Tween20/0.02% NaN₃.

Following this preparation, titration of antibody variants (50 μl,concentrations from 12 nM to 0.12 nM for IL-17A and 1200 nM to 40 nM forIL-17A/F, steps of 3) were pre-incubated with human IL-17A biotin (50 μlat 0.94 nM) or IL-17A/F (50 μl at 31 nM) for 30 minutes at roomtemperature.

100 μl of the mixture were added to the well for 3 hours and 30 minutesat room temperature. After washing with PBS/0.05% Tween20/0.02% NaN₃,four times alkaline phosphatase-conjugated streptavidin was added at afinal dilution of 1/10000 (100 μl/well). After 45 minutes at roomtemperature plates were washed again 4 times with PBS/0.05%Tween20/0.02% NaN₃ and the substrate p-nitrophenylphosphate indiethanolamine buffer pH 9.8 (1 mg/ml), was added (100 μl/well).

Plates were read after 30 minutes in spectra Max M5 Microplate reader,filters 405 and 490 nm (triplicates). The calculation of the percentageof inhibition and IC₅₀ for different antibody variants was done using afour parameter logistic model (Excel Xlfit; FIT model 205).

Results

Data show that both XAB4 and XAB1 are able to block the binding ofhuIL-17A and huIL-17A/F to the huIL-17RA. The higher affinity of XAB4for IL-17A and IL-17A/F is reflected in a higher inhibitory capacity.IC₅₀ values are reported in the table. The higher concentrations neededto block the IL-17A/F-IL-17RA interaction are mostly explained by thefact that about 30 fold higher concentrations of IL-17A/F were used inthe assay. The antibody binds to the A subunit of A/F and thereforecannot prevent binding of the F subunit to the IL-17RA. However, bindingof F to IL-17RA is rather weak, in the 300 nM range.

TABLE 26 XAB4 and XAB1 inhibit the binding of huIL-17A and huIL-17A/F tohuIL-17RA. XAB4 XAB1 Control Ligand\Receptor IC50 (nM) IC50 (nM)antibody interaction (mean ± SEM) (mean ± SEM) (nM) huIL-17A\huIL-17RA0.321 ± 0.037 0.830 ± 0.112 >60 huIL-17A/F\huIL-17RA 153.9 ± 18.9  301.3± 51.9 

Example 12. In Vitro Neutralisation of Human IL-17A and IL-17A/FActivity by Antibody Variants of the Disclosure

(i) Assay on C20A4C16 Cells (Human Chondrocyte Cell Line)

C20A4016, or C-20/A4, clone 6, (Goldring M B, et al 1994, J Clin Invest;94:2307-16) cells were cultured in RPMI (Gibco Cat #61870-010)supplemented with 10% fetal calf serum ultra-low IgG (Gibco Cat#16250-078; lot 1074403), β-mercapto ethanol (5×10⁻⁵ M final), andNormocin (0.1 mg/ml, InvivoGen Cat # ant-nr-2).

The cells were detached from plastic using an Accutase solution (PAA Cat# L11-007). Cells were distributed into 96 well microtiter plates at adensity of 5×10³ in 100 μl well in RPMI 1640 (Gibco Cat #61870-010)without fetal calf serum, β-mercaptoethanol (5×10⁻⁵ M final) andNormocin (0.1 mg/ml).

The 020A4016 cells were allowed to adhere to the plates overnight. Thenext morning, different concentrations of recombinant huIL-17A (SEQ IDNO: 76; MW 32000), recombinant huIL-17A/F (MW 32800), recombinanthuIL-17F (SEQ ID NO: 77; MW 30000), or control medium in the presence ofhuman TNFα (Novartis; MW 17500) were added in a volume of 50 μl totriplicate wells in the presence of 50 μl of different concentrations oftest antibody (XAB4; XAB1), control antibody (Simulect® 1.1% solution,Batch C0011, 831179) or control medium to reach the final volume of 200μl/well and the final concentration of 0.5% fetal calf serum.

HuIL-17A (30 μM), huIL-17A/F (300 μM) and huIL-17F (10 nM) were addedtogether with huTNFα (6 pM). XAB4 (MW 150000) was added in aconcentration range from 1 to 0.003 nM to neutralize huIL-17A, in aconcentration range from 10 to 0.03 nM to neutralize huIL-17A/F and in aconcentration range from 3 μM to 30 nM for huIL-17F. XAB1 (MW 150000)was added in a concentration range from 3 to 0.01 nM to neutralizehuIL-17A, in a concentration range from 10 to 0.03 nM to neutralizehuIL-17A/F and in a concentration range from 3 μM to 30 nM for huIL-17F.Simulect® was added in a concentration range between 3 μM to 100 nM.Culture supernatants were collected after an incubation of 24 h andhuIL-6 production was measured by ELISA.

(ii) Assay on BJ Cells (Human Fibroblasts)

BJ cells (human skin fibroblasts from ATCC Cat # CRL 2522) were culturedin RPMI (Gibco Cat #61870-010) supplemented with 10% fetal calf serumultra-low IgG (Gibco Cat #16250-078; lot 1074403), ß-mercaptoethanol(5×10⁻⁵ M final) and Normocin (0.1 mg/ml, InvivoGen Cat # ant-nr-2). Thecells were detached from plastic using an Accutase solution (PAA Cat #L11-007).

The cells were distributed into 96 well microtiter plates at a densityof 5×10³ in 100 μl well in RPMI 1640 without fetal calf serum,ß-mercaptoethanol (5×10⁻⁵ M final) and Normocin (0.1 mg/ml). The BJcells were allowed to adhere to the plates overnight. The next morning,different concentrations of rhuIL-17A (SEQ ID NO: 76; MW 32000),rhuIL-17A/F (MW 32800) and rhuIL-17F (SEQ ID NO: 77; MW 30000), orcontrol medium in the presence of human TNFα (Novartis; MW 17500) wereadded in a volume of 50 μl to triplicate wells in the presence of 50 μlof different concentrations of test antibody (XAB4; XAB1), controlantibody (Simulect® 1.1% solution, Batch # C0011, 831179), or controlmedium to reach the final volume of 200 μl/well and the finalconcentration of 2.5% fetal calf serum.

HuIL-17A (30 μM), huIL-17A/F (300 μM) and huIL-17F (10 nM) were addedtogether with huTNFα (6 μM). XAB4 (MW 150000) was added in aconcentration range from 1 to 0.003 nM to neutralize huIL-17A, in aconcentration range from 10 to 0.03 nM to neutralize huIL-17A/F and in aconcentration range from 3 μM to 30 nM for huIL-17F. XAB1 (MW 150000)was added in a concentration range from 3 to 0.01 nM to neutralizehuIL-17A, in a concentration range from 10 to 0.03 nM to neutralizehuIL-17A/F and in a concentration range from 3 μM to 30 nM for huIL-17F.Simulect® was added in a concentration range between 3 μM to 100 nM.Culture supernatants were collected after an incubation of 24 h andhuIL-6 and huGROα production were measured by ELISA.

(iii) Detection Assays

1) ELISA for Detection of Human IL-6 Production

ELISA microtiter plates were coated with an anti-human IL-6 mouse Mab(R&D Systems® Cat # MAB206, 100 μl/well at 1 μg/ml) in PBS 0.02% NaN₃and incubated overnight at +4° C. The following day, microtiter plateswere blocked with 300 μl of PBS/2% BSA/0.02% NaN₃ for 3h at roomtemperature. Plates were then washed 4 times with PBS/0.05%Tween20/0.02% NaN₃. Culture supernatants of C20A4C16 (final dilution 1:5for cultures stimulated with huIL-17A plus huTNFα, or 1:2 for culturesstimulated with huTNFα plus huIL-17A/F or IL-17F; 100 μl/well) or BJcells (final dilution 1:10 for cultures stimulated with huIL-17A plushuTNFα, or 1:5 for cultures stimulated with huTNFα plus huIL-17A/F orIL-17F; 100 μl/well) were added.

To establish a titration curve, rhuIL-6 (Novartis; 100 μl/well) wastitrated from 500 μg/ml to 7.8 pg/ml in 1:2 dilution steps. After anovernight incubation at room temperature, plates were washed 4 timeswith PBS/0.05% Tween 20/0.02% NaN₃. A biotin-conjugated goat anti-humanIL-6 antibody was added (R&D Systems® Cat # BAF206, 30 ng/ml; 100μl/well). Samples were left to react for 4 h at room temperature. Afterwashing (4 times), alkaline phosphatase-conjugated streptavidin (Jacksonlmmunoresearch Cat #016-050-084) was added at a final dilution of1/10000 (100 μl/well).

After 40 minutes at room temperature, plates were washed again 4 times.P-Nitrophenyl Phosphate substrate tablets (Sigma; 5 mg, Cat # N9389; 20mg, Cat # N2765) were dissolved in diethanolamine buffer pH 9.8 to givea final concentration of 1 mg/ml. 100 μl were added to each well and theO.D. was read after 1 h in a Spectra Max M5 Microplate Reader (MolecularDevices) using filters of 405 and 490 nm.

2) ELISA for Detection of Human GROα Production

ELISA microtiter plates were coated with an anti-human GROα mouse mAb(R&D Systems® Systems® Cat # MAB275, 100 μl/well at 1.5 μg/ml) inPBS/0.02% NaN₃ and incubated overnight at 4° C. The following day,microtiter plates were blocked with 300 μl of PBS/2% BSA/0.02% NaN₃ for3 h at room temperature. Plates were then washed 4 times with PBS/0.05%Tween20/0.02% NaN₃. Culture supernatants of BJ cells (final dilution1:2; 100 μl/well) were added.

To establish a titration curve, human GROα (R&D Systems® Cat #275-GR/CF;100 μl/well) was titrated from 2 ng/ml to 0.03 ng/ml in 1:2 dilutionsteps.) After an overnight incubation at room temperature, plates werewashed 4 times with PBS/0.05% Tween 20/0.02% NaN₃.

A biotin-conjugated goat anti-human GROα antibody was added (R&DSystems® Cat # BAF275; 100 ng/ml; 100 μl/well). Samples were left toreact for 4 h at room temperature. After washing (4 times), alkalinephosphatase-conjugated streptavidin (Jackson lmmunoresearch Cat#016-050-084) was added at a final dilution of 1/10000 (100 μl/well).After 40 minutes at room temperature, plates were washed again 4 times.P-Nitrophenyl Phosphate substrate tablets (Sigma; 5 mg Cat # N9389; 20mg, Cat # N2765) were dissolved in diethanolamine buffer pH 9.8 to givea final concentration of 1 mg/ml. 100 μl were added to each well and theO.D. was read after 1 h in a Spectra Max M5 Microplate Reader (MolecularDevices) using filters of 405 and 490 nm.

3) Calculations

Data are reported as Means+/−SEM. Four parameter curve fitting was usedfor ELISA calculations. IC₅₀ values for inhibition of IL-6 and GRO-αsecretion by antibodies were calculated using Xlfit (FIT model 205).

(iv) Results

1) Assay on C20A4C16 Cells (Human Chondrocyte Cell Line)

Both XAB4 and XAB1 are able to neutralize the induction of huIL-6secretion by C20A4C16 cells stimulated with rhuIL-17A and rhuIL-17A/F inthe presence of rhuTNFα. Control antibody (Simulect®) at 100 nM has noeffect. IC₅₀ values (means±SEM) for XAB4 and XAB1 are reported in

Table 27. No inhibition on huIL-17F is observed even at Abconcentrations of 3 μM.

TABLE 27 Inhibitory effects of XAB4 and XAB1 on huIL-6 secretion byC20A4CI6 cells. XAB4 XAB1 Control IC50 (nM) IC50 (nM) antibody Stimuli(means ± SEM) (means ± SEM) (nM) rhuIL-17A (1 nM)^(a) 0.44 ± 0.06 >100rhuIL-17A/F (3 nM)^(a) 1.30 ± 0.18 >100 rhuIL-17F (30nM)^(a) >3000 >1000 rhuIL-17A (30 pM) + 0.024 ± 0.004 1.21 ± 0.09 >3000rhuTNF-α (6 pM)^(b) rhuIL-17A/F (300 0.108 ± 0.02  >10 >3000 pM) +rhuTNF-α (6 pM)^(b) rhuIL-17F (10 nM) + >3000 >3000 >3000 rhuTNF-α (6pM)^(b) ^(a)Background of hu IL-6 production without stimulation (0.13 ±0.003) is subtracted ^(b)Background of huIL-6 production in cultureswith TNF alone (0.20 ± 0.003) is subtracted

From these experiments it is evident that the parental XAB1 antibodyshares neutralizing activity with its derivatives. The XAB4 variant isalso seen to have a higher neutralizing activity than XAB1.

In an additional experiment, analogous to the experiment describedabove, all the antibodies XAB1-XAB5 were compared, as seen in

Table 28. Here it can be seen that the inhibition profiles for XAB2,XAB3 and XAB5 are comparable to those observed for XAB4 and XAB1,especially to XAB4.

TABLE 28 Table Inhibitory effects of XAB antibodies on huIL-6 secretionby C20A4CI6 cells. XAB1 XAB2 XAB3 XAB4 XAB5 IC50 (nM) IC50 (nM) IC50(nM) IC50 (nM) IC50 (nM) Stimuli Means ± SEM Means ± SEM Means ± SEMMeans ± SEM Means ± SEM rhuIL-17A 0.29 ± 0.03 0.72 ± 0.08 0.63 ± 0.150.51 ± 0.04 0.55 ± 0.01 (0.5 nM) ^(a) ^(a) Background of HuIL-6production without stimuli (0.04 ± 1.13 ng/ml) is subtracted.

2) Assay on BJ Cells (Human Fibroblasts)

Both XAB4 and XAB1 neutralize the induction of huIL-6 and huGROαsecretion by BJ cells stimulated with rhuIL-17A and rhuIL-17A/F in thepresence of huTNFα. Control antibody (Simulect®) at 100 nM has noeffect. IC₅₀ values for inhibition of IL-6 and hu GROα are reported in

Table 29 and Table 30. Inhibition on huIL-17F is not observed even at Abconcentrations of 3 μM. From these experiments it is evident that theparental XAB1 antibody shares neutralizing activity with itsderivatives.

The XAB4 variant is also seen to have a higher neutralizing activitythan XAB1.

TABLE 29 Inhibitory effect of XAB4 and XAB1 on huIL-6 secretion by BJcells. XAB4 XAB1 Control IC50 (nM) IC50 (nM) antibody Stimuli Means ±SEM Means ± SEM (nM) rhuIL-17A (1 nM)^(a) 0.63 ± 0.02 >100 rhuIL-17A/F(3 nM)^(a) 1.68 ± 0.05 >100 rhuIL-17F (30 nM)^(a) >3000 >1000 rhuIL-17A(30 pM) + 0.012 ± 0.002 0.47 ± 0.02 >3000 rhuTNF-α (6 pM)^(b)rhuIL-17A/F (300 pM) + 0.17 ± 0.01 3.83 ± 0.63 >3000 rhuTNF-α (6 pM)^(b)rhuIL-17F (10 nM) + >3000 >3000 >3000 rhuTNF-α (6 pM)^(b) ^(a)Backgroundof hu IL-6 production without stimuli (0.32 ± 0.002 ng/ml) issubtracted. ^(b)Background of huIL-6 production in cultures stimulatedwith TNF alone (0.45 ± 0.02 ng/ml) is subtracted.

TABLE 30 Inhibitory effect of XAB4 and XAB1 on hu-GRO-alpha secretion byBJ cells. XAB4 XAB1 Control IC50 (nM) IC50 (nM) antibody Stimuli Means ±SEM Means ± SEM (nM) IL-17A (1 nM) ^(a) 0.35 ± 0.01  >100 IL-17A/F (3nM) ^(a) 1.11 ± 0.05  >100 IL-17F (30 nM) ^(a) >3000 >1000 IL-17A (30pM) + 0.007 ± 0.0004 0.72 ± 0.12 >3000 TNF-α (6 pM) ^(b) IL-17A/F (300pM) + 0.1 ± 0.01 6.22 ± 0.44 >3000 TNF-α (6 pM) ^(b) IL-17F (10nM) + >3000 >3000 >3000 TNF-α (6 pM) ^(b) ^(a) Background of hu GROαproduction without stimuli (0.03 ± 0.01 ng/ml) is subtracted. ^(b)Background of hu GROα production in cultures with TNF alone (0.15 ±0.008 ng/ml) is subtracted.

In additional experiments, analogous to the experiments described above,all the antibodies XAB1-XAB5 were compared, as seen in Table 31 andTable 32. Here it can be seen that the inhibition profiles for XAB2,XAB3 and XAB5 are comparable to those observed for XAB4 and XAB1,especially to XAB4.

TABLE 31 Inhibitory effects of XAB antibodies on huIL-6 secretion by BJcells. XAB1 XAB2 XAB3 XAB4 XAB5 IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM)IC50 (nM) Stimuli Means ± SEM Means ± SEM Means ± SEM Means ± SEM Means± SEM rhuIL-17A 4.97 ± 0.59 0.64 ± 0.22 0.50 ± 0.002 0.55 ± 0.04 0.54 ±0.02 (0.5 nM) ^(a) ^(a) Background of HuIL-6 production without stimuli(0.15 ± 4.06 ng/ml) is subtracted

TABLE 32 Inhibitory effects of XAB antibodies on huGROα secretion by BJcells. XAB1 XAB2 XAB3 XAB4 XAB5 IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM)IC50 (nM) Stimuli Means ± SEM Means ± SEM Means ± SEM Means ± SEM Means± SEM rhuIL-17A 1.39 ± 0.07 0.40 ± 0.06 0.42 ± 0.01 0.44 ± 0.04 0.46 ±0.05 (0.5 nM) ^(a) ^(a) Background of HuGROα production without stimuli(0.03 ± 0.02 ng/ml) is subtracted

Example 13. In Vitro Neutralization of Mouse IL-17A and IL-17A/FActivity by Antibody Variants of the Disclosure

CMT-93 cells (ATCC CCL-223) were cultured in RPMI (Gibco Cat #61870-010)supplemented with 10% fetal calf serum ultra-low IgG (Gibco Cat#16250-078; lot 1074403), β-mercaptoethanol (5×10⁻⁵M final) and Normocin(0.1 mg/ml, InvivoGen Cat # ant-nr-2).

The cells were detached from plastic using an Accutase solution (PAA Cat# L11-007) and distributed into 96 wells microtiter plates at a densityof 5×10³ in 100 μl well in RPMI 1640 without fetal calf serum,ß-mercaptoethanol and normocin.

The cells were allowed to adhere to the plates overnight. The nextmorning, rmIL-17A (SEQ ID NO: 83, MW 31000) at 1 nM, rmIL-17A/F (R&DSystems® Cat #5390-IL; MW 30400) at 3 nM, rmIL-17F (SEQ ID NO: 84; MW30000) at 30 nM, rratIL-17A (SEQ ID NO: 85; MW 31000) at 1 nM or controlmedium were added in a volume of 50 μl to triplicate wells in thepresence of 50 μl of different concentrations of test antibodies (XAB4or XAB1), control antibodies (Simulect® 1.1% solution; C0011, 831179) orcontrol medium to reach the final volume of 200 μl/well and the finalconcentration of 1% fetal calf serum.

Culture supernatants were collected after an incubation of 24 h and KCproduction was measured by ELISA.

(i) ELISA for Detection of Mouse KC Production

ELISA microtiter plates were coated with a rat anti-mouse KC MAb (R&DSystems® Cat # MAB453; 100 μl/well at 1 μg/ml) in PBS/0.02% NaN₃ andincubated overnight at 4° C. The following day, microtiter plates wereblocked with 300 μl of PBS/2% BSA/0.02% NaN₃ for 3 h at roomtemperature. Plates were then washed 4 times with PBS/0.05%Tween20/0.02% NaN₃. Culture supernatants of CMT-93 cells (final dilution1:5; 100 μl/well) were added.

To establish a titration curve, mouse KC (R&D Systems® #453-KC, 100μl/well) was titrated from 1 ng/ml to 0.016 ng/ml in 1:2 dilution steps.After an overnight incubation at room temperature, plates were washed 4times with PBS/0.05% Tween 20/0.02% NaN₃. A biotin-conjugated goatanti-mouse KC antibody (R&D Systems® Cat # BAF453; 100 μl/well) at 0.1μg/ml was added. Samples were left to react for 4 h at room temperature.After washing (4 times), alkaline phosphatase-conjugated streptavidin(Jackson lmmunoresearch Cat #016-050-084) was added at a final dilutionof 1/10000 (100 μl/well). After 40 minutes at room temperature, plateswere washed again 4 times. P-Nitrophenyl Phosphate substrate tablets(Sigma; 5 mg Cat # N9389; 20 mg Cat # N2765) were dissolved indiethanolamine buffer pH 9.8 to give a final concentration of 1 mg/ml.100 μl culture supernatants were added to each well and the O.D. wasread after 1 h in a Spectra Max M5 Microplate Reader (Molecular Devices)using filters of 405 and 490 nm.

(ii) Calculations

Data are reported as Means+/−SEM. Four parameter curve fitting was usedfor ELISA calculations. IC₅₀ values for inhibition of KC secretion byantibodies were calculated using Xlfit™ (FIT model 205).

(iii) Results

Both XAB4 and XAB1 are able to neutralize the induction of mouse KCsecretion by CMT-93 cells stimulated with mouse or rat IL-17A and mouseIL-17A/F. Control antibody (Simulect®) has no effect. IC₅₀ values(means±SEM) for XAB4 and XAB1 are reported in Table 33. Inhibition onhuIL-17F is not observed even at Ab concentrations of 10 μM.

TABLE 33 Inhibitory effect of XAB4 and XAB1 on mouse KC secretion byCMT-93 cells. XAB4 XAB1 Control IC50 (nM) IC50 (nM) antibody StimuliMeans ± SEM Means ± SEM (nM) mIL-17A (1 nM) ^(a) 13.8 ± 0.48 539 ±29.4 >3000 mIL-17A/F (3 nM) ^(a) 10.3 ± 1.06 >1000  >3000 mIL-17F (30nM) ^(a) >10000 >10000 >3000 rIL-17A (1 nM) ^(a)  6.7 ± 0.84 467 ±25.1 >3000 ^(a) Background of KC production without stimuli (0.07 ±0.001 ng/ml) is subtracted.

From these experiments it is evident that both the parental XAB1antibody, as well as its derivates, has neutralizing activity. The XAB4variant is also seen to have a higher neutralizing activity than XAB1.

In an additional experiment, analogous to the experiment describedabove, all the antibodies XAB1-XAB5 were compared, as seen in Table 34.Here it can be seen that the inhibition profiles for XAB2, XAB3 and XAB5are comparable to those observed for XAB4 and XAB1, especially to XAB4.

TABLE 34 Inhibitory effects of XAB antibodies on KC secretion by CMT-93cells. XAB1 XAB2 XAB3 XAB4 XAB5 IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM)IC50 (nM) Stimuli Means ± SEM Means ± SEM Means ± SEM Means ± SEM Means± SEM mIL-17A 128 ± 14.2 20.9 ± 0.96 <1 7.0 ± 0.29 7.8 ± 0.78 (0.15 nM)^(a) ^(a) Background of KC production without stimuli (0.19 ± 5.81ng/ml) is subtracted.

Example 14. Rat Antigen-Induced Arthritis Assay (Rat AIA)

Female Lewis rats (120-150 g) were sensitized intradermally on the backat two sites to methylated bovine serum albumin (mBSA) homogenized 1:1with complete Freund's adjuvant on days −21 and −14 (0.1 ml containing 5mg/ml mBSA). On day 0, the rats were anaesthetized using a 5%isoflurane/air mixture and maintained using isoflurane at 3.5% via aface mask for the intra-articular injections. The right knee received 50μl of 10 mg/ml mBSA in 5% glucose solution (antigen injected knee),while the left knee received 50 μl of 5% glucose solution alone (vehicleinjected knee). The diameters of the left and right knees were thenmeasured using calipers immediately after the intra-articular injectionsand again on days 2, 4, and 7.

Treatments were administered by single subcutaneous injection on day −3.The antibody of the disclosure was injected at 0.15, 1.5, 15 and 116mg/kg. Right knee swelling was calculated as a ratio of left kneeswelling, and the R/L knee swelling ratio plotted against time to giveArea Under the Curve (AUC) graphs for control and treatment groups. Thepercentage inhibitions of the individual animals in each treatment groupAUCs were calculated vs. the control group AUC (0% inhibition) using anExcel spreadsheet.

Results

Results are shown in Table 35. Dose related inhibition of right kneeswelling was demonstrated for XAB4 with a calculated ED₅₀ of 1.68 mg/kgs.c.

TABLE 35 Effects of single dose treatment with XAB4 on knee swellingfrom day 0 to day 7 in Lewis rat antigen-induced arthritis. Antibodydose (mg/kg) Percentage inhibition of knee swelling AUC 0.15 18.46 ±1.61*  1.5 65.76 ± 3.41** 15 71.59 ± 1.27** 116 77.01 ± 1.72** Datapoints represent the means ± SEM of n = 5 animals. *p < 0.05 and **p <0.01 ANOVA followed by Dunnett's test vs Control curve.

Similarly, dose related inhibition of knee swelling was demonstrated forXAB4 in a model using Wistar rats (data not shown), and in a model usingmouse antigen-induced arthritis model (data not shown).

Example 15. Angiogenesis Mechanistic Model

Chambers containing human IL-17A (between 150 and 200 ng), when placedsubcutaneously in a mouse, cause new blood vessel growth around theimplant. The amount of angiogenesis correlates with the weight of newlyformed tissue in this area. Prophylactic treatment with XAB4 at 0.01,0.03, 0.1, 0.3, 1 and 3 mg/kg inhibited human IL-17 inducedangiogenesis. The 5 higher doses all led to a potent and significantinhibition of tissue chamber weight. The 4 higher doses showed no dosedependency, however, the dose of 0.03 mg/kg was less efficacious thandoses of 0.1 mg/kg and above. This study demonstrates that the potentangiogenic effect of IL-17A can be neutralized with an anti-IL-17Aantibody and provides experimental evidence of the effectiveness of XAB4for human IL-17A in vivo.

Example 16. Experimental Autoimmune Encephalomyelitis (EAE) Model

The experimental autoimmune encephalomyelitis (EAE) model is a knownanimal model for multiple sclerosis (reviewed e.g. in Constantinescu etal., Br J Pharmacol 2011). It has been shown that inhibition of IL-17reduces EAE severity in C57131/6 mice (Haak S et al 2009, JCI;119:61-69).

Female C57Bl/6 mice (aged 9 weeks, Harlan, Germany) were immunized witha 50/50 mixture of recombinant rat myelin oligodendrocyte glycoproteinpeptide (MOG₁₋₁₂₅) (generated in-house) and complete Freund's adjuvant(CFA, generated by adding 8 mg/ml Mycobacterium tuberculosis strainH37RA (Difco) to Incomplete Freund's adjuvant (IFA, Sigma). Immunizationwas performed by subcutaneous injection with 200 μg/animal of MOG₁₋₁₂₅at the base of tail on day 0. In addition, 200 ng/animal pertussis toxin(PT) was injected intraperitoneally on days 0 and 2.

Both therapeutic treatment effect and prophylactic treatment effect ofXAB4 was tested.

Therapeutic Treatment

For the therapeutic treatment 16 mice were used (8 for XAB4 and 8 forcontrol). Treatment was initiated once the animals had a clinical scoreof at least 2.5 (severe hind limb weakness) for 3 days. After this, 15mg/kg XAB4 or isotype control antibody was injected subcutaneously eachweek with a single dose.

The results are shown in FIGS. 11 to 15 (d.p.i is days postimmunization). In all figures, XAB4 is represented by circles and theisotype control is represented by squares. The therapeutic score(mean+SEM) is shown in FIG. 11. It is clearly seen that animals treatedwith XAB4 has a lower mean clinical score than isotype control. FIG. 12shows the weight change (%) for the two groups of mice, and FIG. 13shows the cumulative therapeutic scores. FIGS. 14 and 15 are comparisonsof the therapeutic score pre- and post-treatment. It is clearly seen inall graphs that XAB4 has a therapeutic effect compared to the isotypecontrol. Thus, therapeutic treatment with XAB4 significantly reduced theseverity of EAE.

Prophylactic Treatment

For the prophylactic treatment 19 mice were used (10 for XAB4 and 9 forcontrol). Each animal was treated one day prior to immunization with 15mg/kg XAB4 or isotype control, through a single subcutaneous injection.After this, 15 mg/kg XAB4 or isotype control antibody was injectedsubcutaneously each week with a single dose.

The results are shown in FIGS. 16 to 20 (d.p.i is days postimmunization). In FIGS. 16 to 19, XAB4 is represented by closed circlesand the isotype control is represented by open squares. The prophylacticscore (mean+SEM) is shown in FIG. 16. It is clearly seen that animalstreated with XAB4 has a lower mean clinical score. FIG. 17 shows theweight change (%) for the two groups of mice, and FIG. 18 shows thecumulative prophylactic scores. The maximum prophylactic score is seenin FIG. 19. It is clearly seen in all graphs that XAB4 has an effectcompared to the isotype control. Furthermore, in FIG. 20, where XAB4 isrepresented by a solid line and isotype control is represented by adotted line, it is seen that EAE onset is later for the group of micetreated with XAB4, compared to the group of mice treated with isotypecontrol.

Thus, it is shown that prophylactic treatment with XAB4 significantlydelayed EAE onset and reduced maximal EAE severity.

Example 17. Attenuation of IL17 Å-Induced Levels of IL6, CXCL1, IL-8,GM-CSF, and CCL2 in Human Astrocytes

The effects of XAB4 on the levels of IL-6, CXCL1, IL-8, GM-CSF, and CCL2in astrocytes isolated from the cerebral cortex of the human brain wereinvestigated. Astrocytes release a number of growth factors, cytokinesand chemokines that allow them to regulate cellular communication,migration and survival of neuronal, glial and immune cells. The directcommunication of astrocytic end-feet with endothelial cells also allowsastrocytes to control function of the blood-brain-barrier. Moreover,astrocytes release and uptake neurotransmitters, such as glutamate, atthe synaptic cleft that allow them to regulate synaptic transmission andexcitoxicity. It is significant that astrocytes form scar pathologyafter CNS injury, thus having apparent opposing roles in normalphysiology and pathophysiology. In disease, astrocytes are suggested toplay roles in a range of psychiatric, neurological and neurodegenerativedisorders, where their role in neuroinflammation is likely to beimportant.

The data showed that co-stimulation with IL-17A and TNFα enhanced therelease of IL-6, CXCL1, IL-8, GM-CSF, and CCL2, and that XAB4 inhibitedlevels of IL-6, CXCL1, IL-8, GM-CSF, and CCL2 in human astrocytes. Thesedata indicate a dominate role for IL-17A in cytokine release fromastrocytes and support their use as drug targets for neuroinflammatorydiseases. It is noteworthy that the pretreatment of human astrocyteswith XAB4 inhibited IL-17A-induced and IL-17A/TNFα-induced, withoutaffecting TNFα-induced, levels of IL-6, CXCL1, IL-8, GM-CSF, and CCL2.Taken together, the data suggested that selective inhibition of IL-17Asignaling with XAB4 attenuates the level of pro-inflammatory cytokinesin human astrocytes. In disease, astrocytes are suggested to play rolesin a range of psychiatric, neurological and neurodegenerative disorders,where their role in neuroinflammation is likely to be important. Noveldrugs that alter astrocyte function are thus of potential value, whereregulation of astrocyte function may prove therapeutically useful.Consequently, since XAB4 was shown to have an effect on IL-6, CXCL1,IL-8, GM-CSF, and CCL2 production of astrocytes, it can be concludedthat XAB4 may be a useful therapeutic agent, such as for treatment ofMultiple Sclerosis (MS).

Materials and Methods

All cytokines were purchased from R&D Systems. Basiliximab (Novartis,Basel, Switzerland) was used as isotype control. Primary antibodies usedwere: anti-IL17RA Alexa Fluor 647 (BG/hIL17AR, Biolegend), anti-IL17RCAlexa Fluor 488 (309822, R&D Systems, UK), anti-p65 (Santa Cruz, USA),mouse IgG Alexa Fluor 647 (MOPC-21, Biolegend, UK), mouse IgG AlexaFluor 488 (133303, R&D System, UK), mouse IgG Biotin (G155-178, BDBiosciences, Switzerland) and rat IgG PE (A95-1, BD Biosciences,Switzerland). Secondary antibodies and dyes used were: biotinylated goatanti-rabbit IgG (BA1000, Vector, UK), streptavidin conjugated AlexaFluor 488 and Alexa Fluor 633 (S11223 and S2137, Life Technology, USA),goat anti-mouse Alexa Fluor 488 and Alexa Fluor 633 (A1101 and A21050,Life Technology, USA), streptavidin BV421 (405226, Biolegend, UK),Hoechst 34580 (H21486, Life Technology, USA).

Human astrocytes derived from cerebral cortex were purchased fromScienCell Research Laboratory (USA) (catalogue number 1800). Cells weregrown as per provider's instructions. Briefly cells were grown in humanastrocyte media (ScienCell catalogue number 1801) supplemented with 1%astrocyte growth supplement (ScienCell catalogue number 1852), 5% fetalcalf serum (ScienCell catalogue number 0010) and 1%Penicillin/Streptomycin (ScienCell catalogue number 0503). Cells weremaintained in T75 culture flasks at 5% CO₂ and 37° C. with the mediachanged every three days until 80% confluent. For all treatments, 70,000cells well plated in 24-well plates, grown for 3 days, serum starved for2-4 hr, after which astrocytes were treated for 2 hr with XAB4, andthereafter treated for 18-20 hr with recombinant human cytokines asindicated in the figure legends. The cell pellets were used to quantifymRNA levels of cytokines by qPCR and the supernatants were used toquantify the protein levels of cytokines by HTRF (Cisbio, France, usedfor IL-6, IL-8 & CXCL1) or AlphaLISA (PerkinElmer, USA, used for CCL2 &GM-CSF).

Measurement of cytokine mRNA was performed by real time-polymerase chainreaction (RT-PCR). Briefly, astrocytes were lysed for 5 min at roomtemperature by gently shaking in 350 μl lysis buffer (RLT buffer with 1%β-mercaptoethanol) and total RNA was extracted using RNeasy Microkit(74004, Qiagen, Switzerland). The cDNA was synthesized using SuperScriptIII reverse transcriptase (18080-400, Life Technology, Switzerland). Theexpression level of each gene was assessed by q-PCR in a Viia7 Real-timePCR machine (Life Technology, Switzerland). Taqman probes were purchasedfrom Life Technology, Switzerland. Each sample was analyzed intriplicate and normalized to hypoxanthine-guaninephosphoribosyltransferase (HPRT). Levels of human IL6, IL8, CXCL1protein (ng/ml) in human astrocyte supernatant (10 μl) were assessed byHTRF (IL6: 621L6PEC; IL8: 621L8PEC; CXCL1: 6FGROPEG, Cisbio, France) andthe level of human CCL2 protein (ng/ml) in human astrocyte supernatant(5 p1) was assessed by AlphaLISA human CCL2/MCP1 (AL244C, PerkinElmer,USA). All measurements were performed according to manufacturer'sinstructions.

Cells suspensions of human astrocytes were obtained from adherentcultures using PBS-5 mM EDTA. For extracellular staining cells wereincubated with whole mouse IgG for 10 min at 4° C. in PBS 2% BSA, andthen stained with antibodies for 30 min at 4° C. in PBS 2% BSA. Forintracellular staining, cells were permeabilized with Cytofix/Cytopermsolution (554714, BD Biosciences, Switzerland) for 20 min at 4° C.before incubating with antibodies for 30 min at 4° C. After filtrationthrough 70 μm strainer, cells were acquired on a BDFortessa (BDBiosciences, Switzerland) and data analyzed using FlowJo software (TreeStar Inc., USA).

After compound treatment, cells were washed in PBS (Sigma Aldrich,Germany) followed by fixation in ice-cold 100% methanol for 10 min.Cells were washed 3×5 min in sterile PBS then permeabilized byincubation with 0.2% Triton-X-100 (Sigma Aldrich, Germany) in PBS for 5min at room temperature. Non-reactive sites were blocked overnight at+4° C. with blocking buffer which consisted of 10% normal goat serum(Life Technology, USA) and 2% bovine serum albumin (Sigma Aldrich,Germany) in PBS. The cells were then incubated in primary antibodyovernight at 4° C. The primary antibody was removed and the cells washed3×5 min PBS after which the secondary fluorescent antibody was appliedfor 2 hr at room temperature. The coverslips were then washed 5×5 min inPBS and counter stained with Hoescht 34580 nuclear stain. The coverslipswere finally mounted on microscope slides in VectashieldR mountingmedium (Vector, UK) and the edges of the coverslip sealed with nailvarnish. The cells were imaged using a Zeiss LSM 510 META confocal laserscanning microscope utilizing an Axiovert 200M inverted microscope(Zeiss Ltd, Germany).

Results

Antagonism of TNF-α or IL-17A stimulation, or IL-17A/TNF-αco-stimulation by XAB4 is shown in FIGS. 21 A to 25 A. Antagonism ofIL-1β or IL-17A/1L-1β co-stimulation by XAB4 is shown in FIGS. 21 B to25 B.

FIG. 21 shows antagonistic effect on IL-6 release, FIG. 22 showsantagonistic effect on CXCL1 release, FIG. 23 shows antagonistic effecton IL-8, FIG. 24 shows antagonistic effect on GM-CSF and FIG. 25 showsantagonistic effect on CCL2.

Primary human astrocytes were treated with increasing concentrations ofXAB4 (0.01 nM, 0.1 nM 1 nM and 10 nM), with or without IL-17A (50ng/ml), TNF-α (10 ng/ml), IL-1β, IL-17A/TNF-α and IL-1β/TNF-α. Allconcentrations used are indicated in the figures. The data shown is arepresentative of two experiments for XAB4 0.01 nM, and of threeexperiments for XAB4 0.1 nM, 1 nM and 10 nM. Values shown aremeans±S.E.M.

As seen in FIG. 21A, XAB4 (all concentrations) has an antagonisticeffect, compared to both control and isotype, on release of IL-6 fromastrocytes stimulated with IL-17A, or IL-17A/TNF-α. Concentration ofIL-6 (ng/ml) is represented by the y-axis and concentration of XAB4 isrepresented on the x-axis (0, i.e. control, 0.01 nM, 0.1 nM 1 nM and 10nM) for each dataset, and 10 nM for isotype. The dataset to the leftrelates to unstimulated cells, the next dataset relates to cellsstimulated with TNF-α (10 ng/ml), the next dataset relates to cellsstimulated with IL-17A (50 ng/ml) and the last dataset relates to cellsco-stimulated with TNF-α (10 ng/ml) and IL-17A (50 ng/ml). The lastdataset has about 10 fold higher scale of the y-axis. As seen in FIG.21B, XAB4 (all concentrations) has no antagonistic effect on cellsstimulated with IL-1β (0.1 ng/ml) or co-stimulated with IL-1β (0.1ng/ml) and IL-17A (50 ng/ml), compared to isotype.

As seen in FIG. 22A, XAB4 (all concentrations) has an antagonisticeffect, compared to both control and isotype, on release of CXCL1 fromastrocytes stimulated with IL-17A, or IL-17A/TNF-α. Concentration ofCXCL1 (ng/ml) is represented by the y-axis and concentration of XAB4 isrepresented on the x-axis (0, i.e. control, 0.01 nM, 0.1 nM 1 nM and 10nM) for each dataset, and 10 nM for isotype. The dataset to the leftrelates to unstimulated cells, the next dataset relates to cellsstimulated with TNF-α (10 ng/ml), the next dataset relates to cellsstimulated with IL-17A (50 ng/ml) and the last dataset relates to cellsco-stimulated with TNF-α (10 ng/ml) and IL-17A (50 ng/ml). The lastdataset has about 10 fold higher scale of the y-axis. As seen in FIG.22B, XAB4 (all concentrations) has no antagonistic effect on cellsstimulated with IL-1β (0.1 ng/ml) or co-stimulated with IL-1β (0.1ng/ml) and IL-17A (50 ng/ml), compared to isotype.

As seen in FIG. 23A, XAB4 (all concentrations) has an antagonisticeffect, compared to control, on release of IL-8 from astrocytesstimulated with IL-17A, or IL-17A/TNF-α. Compared to isotype, XAB4 (0.1nM, 1 nM and 10 nM) has an antagonistic effect on release of IL-8.Concentration of IL-8 (ng/ml) is represented by the y-axis andconcentration of XAB4 is represented on the x-axis (0, i.e. control,0.01 nM, 0.1 nM 1 nM and 10 nM) for each dataset, and 10 nM for isotype.The dataset to the left relates to unstimulated cells, the next datasetrelates to cells stimulated with TNF-α (10 ng/ml), the next datasetrelates to cells stimulated with IL-17A (50 ng/ml) and the last datasetrelates to cells co-stimulated with TNF-α (10 ng/ml) and IL-17A (50ng/ml). The last dataset has about 5 fold higher scale of the y-axis. Asseen in FIG. 23B, XAB4 (all concentrations) has no antagonistic effecton cells stimulated with IL-1β (0.1 ng/ml) or co-stimulated with IL-1β(0.1 ng/ml) and IL-17A (50 ng/ml), compared to isotype.

As seen in FIG. 24A, XAB4 (all concentrations) has an antagonisticeffect, compared to both control and isotype, on release of GM-CSF fromastrocytes stimulated with IL-17A/TNF-α. XAB4 (0.1 nM, 1 nM and 10 nM)has an antagonistic effect on release of GM-CSF from astrocytesstimulated with IL-17A, compared to isotype and control. Concentrationof GM-CSF (ng/ml) is represented by the y-axis and concentration of XAB4is represented on the x-axis (0, i.e. control, 0.01 nM, 0.1 nM 1 nM and10 nM) for each dataset, and 10 nM for isotype. The dataset to the leftrelates to unstimulated cells, the next dataset relates to cellsstimulated with TNF-α (10 ng/ml), the next dataset relates to cellsstimulated with IL-17A (50 ng/ml) and the last dataset relates to cellsco-stimulated with TNF-α (10 ng/ml) and IL-17A (50 ng/ml). As seen inFIG. 24B, XAB4 (all concentrations) has no or low antagonistic effect oncells stimulated with IL-1β (0.1 ng/ml) or co-stimulated with IL-1β (0.1ng/ml) and IL-17A (50 ng/ml), compared to isotype.

As seen in FIG. 25A, XAB4 (all concentrations) has an antagonisticeffect, compared to both control and isotype, on release of CCL2 fromastrocytes stimulated with IL-17A. XAB4 (0.1 nM, 1 nM and 10 nM) has anantagonistic effect on release of CCL2 from astrocytes stimulated withIL-17A/TNF-α, compared to isotype and control. Concentration of CCL2(ng/ml) is represented by the y-axis and concentration of XAB4 isrepresented on the x-axis (0, i.e. control, 0.01 nM, 0.1 nM 1 nM and 10nM) for each dataset, and 10 nM for isotype. The dataset to the leftrelates to unstimulated cells, the next dataset relates to cellsstimulated with TNF-α (10 ng/ml), the next dataset relates to cellsstimulated with IL-17A (50 ng/ml) and the last dataset relates to cellsco-stimulated with TNF-α (10 ng/ml) and IL-17A (50 ng/ml). As seen inFIG. 25B, XAB4 (all concentrations) has no antagonistic effect on cellsstimulated with IL-1β (0.1 ng/ml) or co-stimulated with IL-1β (0.1ng/ml) and IL-17A (50 ng/ml), compared to isotype.

Taken together, the data suggested that selective inhibition of IL-17Asignaling with XAB4 attenuates the level of pro-inflammatory cytokinesin human astrocytes. In disease, astrocytes are suggested to play rolesin a range of psychiatric, neurological and neurodegenerative disorders,where their role in neuroinflammation is likely to be important. SinceXAB4 was shown to have an effect on IL-6, CXCL1, IL-8, GM-CSF, and CCL2production of astrocytes, XAB4 may be a useful therapeutic agent, suchas for treatment of Multiple Sclerosis (MS).

Sequence Information

Sequence data relating to XAB1, XAB2, XAB3, XAB4 and XAB5 is summarizedbelow for ease of reference.

Table 1 describes the amino acid sequences (SEQ ID NOs) of the fulllength heavy and light chains of examples XAB1, XAB2, XAB3, XAB4 andXAB5.

The antibodies XAB1, XAB2, XAB3, XAB4 or XAB5 can be produced usingconventional antibody recombinant production and purification processes.For example, the coding sequences as described in Table 3 or Table 4 arecloned into a production vector for recombinant expression in mammalianproduction cell line.

Table 2 summarizes the variable heavy (VH) and light chain (VL) aminoacid sequence of XAB1, XAB2, XAB3, XAB4 or XAB5, which can be used togenerate chimeric antibodies from XAB1, XAB2, XAB3, XAB4 or XAB5.

Table 5 summarizes the useful CDR sequences of XAB1, XAB2, XAB3, XAB4and XAB5 (plus consensus sequences) to generate alternative CDR graftedantibodies, wherein the CDR regions from XAB1, XAB2, XAB3, XAB4 and XAB5are defined according to Kabat definition.

Table 6 summarizes the useful CDR sequences of XAB1, XAB2, XAB3, XAB4and XAB5 (plus consensus sequences) to generate alternative CDR graftedantibodies, wherein the CDR regions from XAB1, XAB2, XAB3, XAB4 and XAB5are defined according to Chothia definition.

All the sequences referred to in this specification (SEQ ID NOs) arefound in Table 36.

Sequence List

Useful amino acids and nucleotide sequences for practicing the inventionare found in Table 36.

TABLE 36 Sequence list Sequence Identifier Antibody/ (SEQ IDAmino acid sequence or polynucleotide sequence Fragment NO:) (PN)XAB1, CDRH1  1 GFTFSSY (CHOTHIA) XAB1, CDRH2  2 KQDGSE (CHOTHIA)XAB1, CDRH3  3 DRGSLYY (CHOTHIA) XAB1, CDRL1  4 SQGIISA (CHOTHIA)XAB1, CDRL2  5 DAS (CHOTHIA) XAB1, CDRL3  6 FNSYPL (CHOTHIA) XAB1, CDRH1 7 SYWMS (KABAT) XAB1, CDRH2  8 NIKQDGSEKYYVDSVKG (KABAT) XAB1, CDRH3  3DRGSLYY (KABAT) XAB1, CDRL1  9 RPSQGIISALA (KABAT) XAB1, CDRL2 10DASSLEN (KABAT) XAB1, CDRL3 11 QQFNSYPLT (KABAT) XAB1, VH 12EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMS WVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRGSLYY WGQGTLVTVSS XAB1, VL 13AIQLTQSPSSLSASVGDRVTITCRPSQGIISALAWYQQKPGKAPKLLIYDASSLENGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKXAB1, HEAVY CHAIN 14 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK XAB1, LIGHT CHAIN 15AIQLTQSPSSLSASVGDRVTITCRPSQGIISALAWYQQKPGKAPKLLIYDASSLENGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC PN ENCODING SEQ 16GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTG ID NO: 12GTGCAGCCTGGCGGCAGCCTGAGACTGAGCTGC GCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAGGCCCCTGGCAAAGGCC TCGAATGGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCC GGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGA GGACACCGCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTACTATTGGGGCCAGGGCACCCT GGTCACCGTGTCCAGC PN ENCODING SEQ 17GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGT ID NO: 13CTGCATCTGTGGGAGACAGAGTCACCATCACTTG CCGGCCAAGTCAGGGCATTATCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGC TCCTGATCTATGATGCCTCCAGTTTGGAAAATGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGA CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAA TAGTTACCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA PN ENCODING SEQ 18 GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTGID NO: 14 GTGCAGCCTGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGA TGTCCTGGGTCCGCCAGGCCCCTGGCAAAGGCCTCGAATGGGTGGCCAACATCAAGCAGGACGGCA GCGAGAAGTACTACGTGGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAG CCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGACCG GGGCAGCCTGTACTATTGGGGCCAGGGCACCCTGGTCACCGTGTCCAGCGCTAGCACCAAGGGCCC CAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCT GGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGT GCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCC AGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGA CAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCT GCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCC CCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGT GGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAG GGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTC TCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGC CCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCT GGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAA CTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGG ACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACC ACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGCAAG PN ENCODING SEQ 19 GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGT ID NO: 15CTGCATCTGTGGGAGACAGAGTCACCATCACTTG CCGGCCAAGTCAGGGCATTATCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGC TCCTGATCTATGATGCCTCCAGTTTGGAAAATGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGA CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAA TAGTTACCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCCGCTCCCAGC GTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGA ACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAG CCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTG AGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCC CCGTGACCAAGAGCTTCAACAGGGGCGAGTGCXAB2, CDRH1  1 GFTFSSY (CHOTHIA) XAB2, CDRH2  2 KQDGSE (CHOTHIA)XAB2, CDRH3  3 DRGSLYY (CHOTHIA) XAB2, CDRL1 20 SQVIISA (CHOTHIA)XAB2, CDRL2  5 DAS (CHOTHIA) XAB2, CDRL3 21 FDSYPL (CHOTHIA) XAB2, CDRH1 7 SYWMS (KABAT) XAB2, CDRH2  8 NIKQDGSEKYYVDSVKG (KABAT) XAB2, CDRH3  3DRGSLYY (KABAT) XAB2, CDRL1 22 RPSQVIISALA (KABAT) XAB2, CDRL2 23DASSLEQ (KABAT) XAB2, CDRL3 24 QQFDSYPLT (KABAT) XAB2, VH 12EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMS WVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRGSLYY WGQGTLVTVSS XAB2, VL 25AIQLTQSPSSLSASVGDRVTITCRPSQVIISALAWYQQKPGKAPKLLIYDASSLEQGVPSRFSGSVSGTDFTL TISSLQPEDFATYYCQQFDSYPLTFGGGTKVEIKXAB2, HEAVY CHAIN 14 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSCLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK XAB2, LIGHT CHAIN 26AIQLTQSPSSLSASVGDRVTITCRPSQVIISALAWYQQKPGKAPKLLIYDASSLEQGVPSRFSGSVSGTDFTLTISSLQPEDFATYYCQQFDSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC PN ENCODING SEQ 16GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTG ID NO: 12GTGCAGCCTGGCGGCAGCCTGAGACTGAGCTGC GCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAGGCCCCTGGCAAAGGCC TCGAATGGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCC GGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGA GGACACCGCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTACTATTGGGGCCAGGGCACCCT GGTCACCGTGTCCAGC PN ENCODING SEQ 27GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTG ID NO: 25AGCGCCAGCGTGGGCGACAGAGTGACCATCACC TGTCGGCCCAGCCAGGTCATCATCAGCGCCCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCC AAGCTGCTGATCTACGACGCCAGCTCCCTGGAACAGGGCGTGCCCAGCCGGTTCAGCGGCAGCGTGT CCGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAG CAGTTCGACAGCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAG PN ENCODING SEQ 18GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTG ID NO: 14GTGCAGCCTGGCGGCAGCCTGAGACTGAGCTGC GCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAGGCCCCTGGCAAAGGCC TCGAATGGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCC GGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGA GGACACCGCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTACTATTGGGGCCAGGGCACCCT GGTCACCGTGTCCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAG CACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGT GTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGG CCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCA ACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGAC CCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCC AAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCC ACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAA GCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGA CTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGA CCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGG AGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGT GGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGAC GGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCA GCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGG CAAG PN ENCODING SEQ 28GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTG ID NO: 26AGCGCCAGCGTGGGCGACAGAGTGACCATCACC TGTCGGCCCAGCCAGGTCATCATCAGCGCCCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCC AAGCTGCTGATCTACGACGCCAGCTCCCTGGAACAGGGCGTGCCCAGCCGGTTCAGCGGCAGCGTGT CCGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAG CAGTTCGACAGCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAGCGTACGGTGGCCG CTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTG CCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGC GGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCC TGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCT GTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC ALTERNATIVE PN 29 GAGGTGCAGCTGGTGGAATCAGGAGGCGACCTGENCODING SEQ ID GTGCAGCCTGGCGGCTCACTGAGACTGAGCTGC NO: 12GCCGCTAGTGGCTTCACCTTTAGTAGCTACTGGA TGAGCTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAGTGGGTGGCCAATATTAAGCAGGACGGCTC AGAGAAGTACTACGTGGACTCAGTGAAGGGCCGGTTCACTATTAGCCGGGATAACGCTAAGAATAGC CTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCTAGAGATAGAGG CTCACTGTACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCT ALTERNATIVE PN 30 GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGAENCODING SEQ ID GCGCTAGTGTGGGCGATAGAGTGACTATCACCTG NO: 25TAGACCTAGTCAGGTGATCATTAGCGCCCTGGCC TGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCTAGTAGTCTGGAACAGG GCGTGCCCTCTAGGTTTAGCGGCTCAGTGTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTGCAG CCCGAGGACTTCGCTACCTACTACTGTCAGCAGTTCGATAGCTACCCCCTGACCTTCGGCGGAGGCAC TAAGGTGGAAATCAAG ALTERNATIVE PN 31GAGGTGCAGCTGGTGGAATCAGGAGGCGACCTG ENCODING SEQ IDGTGCAGCCTGGCGGCTCACTGAGACTGAGCTGC NO: 14GCCGCTAGTGGCTTCACCTTTAGTAGCTACTGGA TGAGCTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAGTGGGTGGCCAATATTAAGCAGGACGGCTC AGAGAAGTACTACGTGGACTCAGTGAAGGGCCGGTTCACTATTAGCCGGGATAACGCTAAGAATAGC CTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCTAGAGATAGAGG CTCACTGTACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCTGCTAGCACCAAGGGCCCAAGTG TCTTTCCCCTGGCCCCCAGCAGCAAGTCCACAAGCGGAGGCACTGCAGCTCTGGGTTGTCTGGTGAA GGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACC TTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTCGTGACTGTGCCTAGTTCCA GCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGA GTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGA GGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGG TGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGG GGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGT GTCCGTCCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACA AGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGT GTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAG GGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAG ACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGT CCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACAC CCAGAAGTCCCTGAGCCTGAGCCCCGGCAAGALTERNATIVE PN 32 GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGA ENCODING SEQ IDGCGCTAGTGTGGGCGATAGAGTGACTATCACCTG NO: 26TAGACCTAGTCAGGTGATCATTAGCGCCCTGGCC TGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCTAGTAGTCTGGAACAGG GCGTGCCCTCTAGGTTTAGCGGCTCAGTGTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTGCAG CCCGAGGACTTCGCTACCTACTACTGTCAGCAGTTCGATAGCTACCCCCTGACCTTCGGCGGAGGCAC TAAGGTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGC TGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCA GTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAA GGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGT ACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGT GC XAB3, CDRH1  1 GFTFSSY (CHOTHIA)XAB3, CDRH2  2 KQDGSE (CHOTHIA) XAB3, CDRH3  3 DRGSLYY (CHOTHIA)XAB3, CDRL1 33 SQGIYWE (CHOTHIA) XAB3, CDRL2  5 DAS (CHOTHIA)XAB3, CDRL3  6 FNSYPL (CHOTHIA) XAB3, CDRH1  7 SYWMS (KABAT) XAB3, CDRH2 8 NIKQDGSEKYYVDSVKG (KABAT) XAB3, CDRH3  3 DRGSLYY (KABAT) XAB3, CDRL134 RPSQGIYWELA (KABAT) XAB3, CDRL2 23 DASSLEQ (KABAT) XAB3, CDRL3 11QQFNSYPLT (KABAT) XAB3, VH 12 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSS XAB3, VL 35 AIQLTQSPSSLSASVGDRVTITCRPSQGIYWELAWYQQKPGKAPKLLIYDASSLEQGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKXAB3, HEAVY CHAIN 14 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSVCLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK XAB3, LIGHT CHAIN 36AIQLTQSPSSLSASVGDRVTITCRPSQGIYWELAWYQQKPGKAPKLLIYDASSLEQGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC PN ENCODING SEQ 16GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTG ID NO: 12GTGCAGCCTGGCGGCAGCCTGAGACTGAGCTGC GCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAGGCCCCTGGCAAAGGCC TCGAATGGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCC GGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGA GGACACCGCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTACTATTGGGGCCAGGGCACCCT GGTCACCGTGTCCAGC PN ENCODING SEQ 37GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTG ID NO: 35AGCGCCAGCGTGGGCGACAGAGTGACCATCACC TGTCGGCCCAGCCAGGGCATCTACTGGGAGCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCC AAGCTGCTGATCTACGACGCCAGCTCCCTGGAACAGGGCGTGCCCAGCCGGTTCAGCGGCAGCGGAT CCGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAG CAGTTCAACAGCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAG PN ENCODING SEQ 18GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTG ID NO: 14GTGCAGCCTGGCGGCAGCCTGAGACTGAGCTGC GCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAGGCCCCTGGCAAAGGCC TCGAATGGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCC GGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGA GGACACCGCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTACTATTGGGGCCAGGGCACCCT GGTCACCGTGTCCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAG CACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGT GTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGG CCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCA ACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGAC CCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCC AAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCC ACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAA GCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGA CTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGA CCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGG AGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGT GGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGAC GGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCA GCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGG CAAG PN ENCODING SEQ 38GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTG ID NO: 36AGCGCCAGCGTGGGCGACAGAGTGACCATCACC TGTCGGCCCAGCCAGGGCATCTACTGGGAGCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCC AAGCTGCTGATCTACGACGCCAGCTCCCTGGAACAGGGCGTGCCCAGCCGGTTCAGCGGCAGCGGAT CCGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAG CAGTTCAACAGCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAGCGTACGGTGGCCG CTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTG CCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGC GGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCC TGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCT GTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC ALTERNATIVE PN 29 GAGGTGCAGCTGGTGGAATCAGGAGGCGACCTGENCODING SEQ ID GTGCAGCCTGGCGGCTCACTGAGACTGAGCTGC NO: 12GCCGCTAGTGGCTTCACCTTTAGTAGCTACTGGA TGAGCTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAGTGGGTGGCCAATATTAAGCAGGACGGCTC AGAGAAGTACTACGTGGACTCAGTGAAGGGCCGGTTCACTATTAGCCGGGATAACGCTAAGAATAGC CTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCTAGAGATAGAGG CTCACTGTACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCT ALTERNATIVE PN 39 GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGAENCODING SEQ ID GCGCTAGTGTGGGCGATAGAGTGACTATCACCTG NO: 35TAGACCTAGCCAGGGAATCTACTGGGAGCTGGCC TGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCTAGTAGTCTGGAACAGG GCGTGCCCTCTAGGTTTAGCGGCTCAGGCTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTGCAG CCCGAGGACTTCGCTACCTACTACTGTCAGCAGTTTAACTCCTACCCCCTGACCTTCGGCGGAGGCAC TAAGGTGGAAATCAAG ALTERNATIVE PN 31GAGGTGCAGCTGGTGGAATCAGGAGGCGACCTG ENCODING SEQ IDGTGCAGCCTGGCGGCTCACTGAGACTGAGCTGC NO: 14GCCGCTAGTGGCTTCACCTTTAGTAGCTACTGGA TGAGCTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAGTGGGTGGCCAATATTAAGCAGGACGGCTC AGAGAAGTACTACGTGGACTCAGTGAAGGGCCGGTTCACTATTAGCCGGGATAACGCTAAGAATAGC CTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCTAGAGATAGAGG CTCACTGTACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCTGCTAGCACCAAGGGCCCAAGTG TCTTTCCCCTGGCCCCCAGCAGCAAGTCCACAAGCGGAGGCACTGCAGCTCTGGGTTGTCTGGTGAA GGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACC TTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTCGTGACTGTGCCTAGTTCCA GCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGA GTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGA GGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGG TGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGG GGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGT GTCCGTCCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACA AGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGT GTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAG GGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAG ACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGT CCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACAC CCAGAAGTCCCTGAGCCTGAGCCCCGGCAAGALTERNATIVE PN 40 GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGA ENCODING SEQ IDGCGCTAGTGTGGGCGATAGAGTGACTATCACCTG NO: 36TAGACCTAGCCAGGGAATCTACTGGGAGCTGGCC TGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCTAGTAGTCTGGAACAGG GCGTGCCCTCTAGGTTTAGCGGCTCAGGCTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTGCAG CCCGAGGACTTCGCTACCTACTACTGTCAGCAGTTTAACTCCTACCCCCTGACCTTCGGCGGAGGCAC TAAGGTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGC TGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCA GTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAA GGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGT ACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGT GC XAB4, CDRH1  1 GFTFSSY (CHOTHIA)XAB4, CDRH2  2 KQDGSE (CHOTHIA) XAB4, CDRH3  3 DRGSLYY (CHOTHIA)XAB4, CDRL1 41 SQGINWE (CHOTHIA) XAB4, CDRL2  5 DAS (CHOTHIA)XAB4, CDRL3  6 FNSYPL (CHOTHIA) XAB4, CDRH1  7 SYWMS (KABAT) XAB4, CDRH2 8 NIKQDGSEKYYVDSVKG (KABAT) XAB4, CDRH3  3 DRGSLYY (KABAT) XAB4, CDRL142 RPSQGINWELA (KABAT) XAB4, CDRL2 23 DASSLEQ (KABAT) XAB4, CDRL3 11QQFNSYPLT (KABAT) XAB4, VH 12 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSS XAB4, VL 43 AIQLTQSPSSLSASVGDRVTITCRPSQGINWELAWYQQKPGKAPKLLIYDASSLEQGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKXAB4, HEAVY CHAIN 14 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSCLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK XAB4, LIGHT CHAIN 44AIQLTQSPSSLSASVGDRVTITCRPSQGINWELAWYQQKPGKAPKLLIYDASSLEQGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC PN ENCODING SEQ 16GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTG ID NO: 12GTGCAGCCTGGCGGCAGCCTGAGACTGAGCTGC GCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAGGCCCCTGGCAAAGGCC TCGAATGGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCC GGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGA GGACACCGCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTACTATTGGGGCCAGGGCACCCT GGTCACCGTGTCCAGC PN ENCODING SEQ 45GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTG ID NO: 43AGCGCCAGCGTGGGCGACAGAGTGACCATCACC TGTCGGCCCAGCCAGGGCATCAACTGGGAGCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCC AAGCTGCTGATCTACGACGCCAGCTCCCTGGAACAGGGCGTGCCCAGCCGGTTCAGCGGCAGCGGAT CCGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAG CAGTTCAACAGCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAG PN ENCODING SEQ 18GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTG ID NO: 14GTGCAGCCTGGCGGCAGCCTGAGACTGAGCTGC GCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAGGCCCCTGGCAAAGGCC TCGAATGGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCC GGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGA GGACACCGCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTACTATTGGGGCCAGGGCACCCT GGTCACCGTGTCCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAG CACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGT GTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGG CCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCA ACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGAC CCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCC AAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCC ACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAA GCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGA CTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGA CCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGG AGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGT GGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGAC GGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCA GCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGG CAAG PN ENCODING SEQ 46GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTG ID NO: 44AGCGCCAGCGTGGGCGACAGAGTGACCATCACC TGTCGGCCCAGCCAGGGCATCAACTGGGAGCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCC AAGCTGCTGATCTACGACGCCAGCTCCCTGGAACAGGGCGTGCCCAGCCGGTTCAGCGGCAGCGGAT CCGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAG CAGTTCAACAGCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAGCGTACGGTGGCCG CTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTG CCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGC GGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCC TGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCT GTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC ALTERNATIVE PN 29 GAGGTGCAGCTGGTGGAATCAGGAGGCGACCTGENCODING SEQ ID GTGCAGCCTGGCGGCTCACTGAGACTGAGCTGC NO: 12GCCGCTAGTGGCTTCACCTTTAGTAGCTACTGGA TGAGCTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAGTGGGTGGCCAATATTAAGCAGGACGGCTC AGAGAAGTACTACGTGGACTCAGTGAAGGGCCGGTTCACTATTAGCCGGGATAACGCTAAGAATAGC CTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCTAGAGATAGAGG CTCACTGTACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCT ALTERNATIVE PN 47 GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGAENCODING SEQ ID GCGCTAGTGTGGGCGATAGAGTGACTATCACCTG NO: 43TAGACCTAGTCAGGGGATTAACTGGGAGCTGGCC TGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCTAGTAGTCTGGAACAGG GCGTGCCCTCTAGGTTTAGCGGCTCAGGCTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTGCAG CCCGAGGACTTCGCTACCTACTACTGTCAGCAGTTTAACTCCTACCCCCTGACCTTCGGCGGAGGCAC TAAGGTGGAAATCAAG ALTERNATIVE PN 31GAGGTGCAGCTGGTGGAATCAGGAGGCGACCTG ENCODING SEQ IDGTGCAGCCTGGCGGCTCACTGAGACTGAGCTGC NO: 14GCCGCTAGTGGCTTCACCTTTAGTAGCTACTGGA TGAGCTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAGTGGGTGGCCAATATTAAGCAGGACGGCTC AGAGAAGTACTACGTGGACTCAGTGAAGGGCCGGTTCACTATTAGCCGGGATAACGCTAAGAATAGC CTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCTAGAGATAGAGG CTCACTGTACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCTGCTAGCACCAAGGGCCCAAGTG TCTTTCCCCTGGCCCCCAGCAGCAAGTCCACAAGCGGAGGCACTGCAGCTCTGGGTTGTCTGGTGAA GGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACC TTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTCGTGACTGTGCCTAGTTCCA GCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGA GTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGA GGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGG TGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGG GGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGT GTCCGTCCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACA AGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGT GTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAG GGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAG ACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGT CCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACAC CCAGAAGTCCCTGAGCCTGAGCCCCGGCAAGALTERNATIVE PN 48 GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGA ENCODING SEQ IDGCGCTAGTGTGGGCGATAGAGTGACTATCACCTG NO: 44TAGACCTAGTCAGGGGATTAACTGGGAGCTGGCC TGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCTAGTAGTCTGGAACAGG GCGTGCCCTCTAGGTTTAGCGGCTCAGGCTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTGCAG CCCGAGGACTTCGCTACCTACTACTGTCAGCAGTTTAACTCCTACCCCCTGACCTTCGGCGGAGGCAC TAAGGTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGC TGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCA GTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAA GGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGT ACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGT GC SECOND 49GAGGTGCAGCTGGTGGAATCTGGCGGCGACCTG ALTERNATIVE PNGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGCG ENCODING SEQ IDCCGCCTCCGGCTTCACCTTCTCCAGCTACTGGAT NO: 12GTCCTGGGTGCGACAGGCCCCTGGCAAGGGACT GGAATGGGTGGCCAACATCAAGCAGGACGGCTCCGAGAAGTACTACGTGGACTCCGTGAAGGGCCG GTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAG GACACCGCCGTGTACTACTGCGCCAGGGACCGGGGCTCCCTGTACTATTGGGGCCAGGGCACCCTG GTGACAGTGTCCTCC SECOND 50GCCATCCAGCTGACCCAGTCCCCCTCCAGCCTGT ALTERNATIVE PNCTGCCTCCGTGGGCGACAGAGTGACCATCACCTG ENCODING SEQ IDTCGGCCCTCCCAGGGCATCAACTGGGAACTGGC NO: 43CTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAA GCTGCTGATCTACGACGCCAGCTCCCTGGAACAGGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCCG GCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAG TTCAACTCCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAG SECOND 51 GAGGTGCAGCTGGTGGAATCTGGCGGCGACCTGALTERNATIVE PN GTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGCG ENCODING SEQ IDCCGCCTCCGGCTTCACCTTCTCCAGCTACTGGAT NO: 14GTCCTGGGTGCGACAGGCCCCTGGCAAGGGACT GGAATGGGTGGCCAACATCAAGCAGGACGGCTCCGAGAAGTACTACGTGGACTCCGTGAAGGGCCG GTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAG GACACCGCCGTGTACTACTGCGCCAGGGACCGGGGCTCCCTGTACTATTGGGGCCAGGGCACCCTG GTGACAGTGTCCTCCGCCTCCACCAAGGGCCCAAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCA CCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTC CTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCT GTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAAC GTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGTGACAAGACCC ACACCTGCCCCCCCTGCCCAGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAA GCCCAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAC GAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGC CCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTG GCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCAGCCCCAATCGAAAAGACCA TCAGCAAGGCCAAGGGCCAGCCAAGAGAGCCCCAGGTGTACACCCTGCCACCCAGCAGGGAGGAGA TGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCAAGCGACATCGCCGTGGA GTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGG CAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCT GCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCAGGCAA G SECOND 52GCCATCCAGCTGACCCAGTCCCCCTCCAGCCTGT ALTERNATIVE PNCTGCCTCCGTGGGCGACAGAGTGACCATCACCTG ENCODING SEQ IDTCGGCCCTCCCAGGGCATCAACTGGGAACTGGC NO: 44CTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAA GCTGCTGATCTACGACGCCAGCTCCCTGGAACAGGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCCG GCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAG TTCAACTCCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAGCGTACGGTGGCCGCTC CCAGCGTGTTCATCTTCCCCCCAAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCT GCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGC AACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGA CCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTC CAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC XAB5, CDRH1  1 GFTFSSY (CHOTHIA) XAB5, CDRH2  2 KQDGSE (CHOTHIA)XAB5, CDRH3  3 DRGSLYY (CHOTHIA) XAB5, CDRL1 41 SQGINWE (CHOTHIA)XAB5, CDRL2  5 DAS (CHOTHIA) XAB5, CDRL3  6 FNSYPL (CHOTH IA)XAB5, CDRH1  7 SYWMS (KABAT) XAB5, CDRH2  8 NIKQDGSEKYYVDSVKG (KABAT)XAB5, CDRH3  3 DRGSLYY (KABAT) XAB5, CDRL1 42 RPSQGINWELA (KABAT)XAB5, CDRL2 10 DASSLEN (KABAT) XAB5, CDRL3 11 QQFNSYPLT (KABAT) XAB5, VH12 EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSS XAB5, VL 53 AIQLTQSPSSLSASVGDRVTITCRPSQGINWELAWYQQKPGKAPKLLIYDASSLENGVPSRFSGSGSGTDFTVLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK XAB5, HEAVY CHAIN 14EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMS WVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRGSLYY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SCLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK XAB5, LIGHT CHAIN 54AIQLTQSPSSLSASVGDRVTITCRPSQGINWELAWYQQKPGKAPKLLIYDASSLENGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC PN ENCODING SEQ 16GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTG ID NO: 12GTGCAGCCTGGCGGCAGCCTGAGACTGAGCTGC GCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAGGCCCCTGGCAAAGGCC TCGAATGGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCC GGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGA GGACACCGCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTACTATTGGGGCCAGGGCACCCT GGTCACCGTGTCCAGC PN ENCODING SEQ 55GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTG ID NO: 53AGCGCCAGCGTGGGCGACAGAGTGACCATCACC TGTCGGCCCAGCCAGGGCATCAACTGGGAGCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCC AAGCTGCTGATCTACGACGCCAGCTCCCTGGAAAACGGCGTGCCCAGCCGGTTCAGCGGCAGCGGAT CCGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAG CAGTTCAACAGCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAG PN ENCODING SEQ 18GAGGTGCAGCTGGTCGAGTCTGGCGGCGACCTG ID NO: 14GTGCAGCCTGGCGGCAGCCTGAGACTGAGCTGC GCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGTCCTGGGTCCGCCAGGCCCCTGGCAAAGGCC TCGAATGGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCC GGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGA GGACACCGCCGTGTACTACTGCGCCAGGGACCGGGGCAGCCTGTACTATTGGGGCCAGGGCACCCT GGTCACCGTGTCCAGCGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAG CACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGT GTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGG CCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCA ACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGAC CCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCC AAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCC ACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAA GCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGA CTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGA CCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGG AGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGT GGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGAC GGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCA GCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGG CAAG PN ENCODING SEQ 56GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTG ID NO: 54AGCGCCAGCGTGGGCGACAGAGTGACCATCACC TGTCGGCCCAGCCAGGGCATCAACTGGGAGCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCC AAGCTGCTGATCTACGACGCCAGCTCCCTGGAAAACGGCGTGCCCAGCCGGTTCAGCGGCAGCGGAT CCGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAG CAGTTCAACAGCTACCCCCTGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAGCGTACGGTGGCCG CTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTG CCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGC GGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCC TGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCT GTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC ALTERNATIVE PN 29 GAGGTGCAGCTGGTGGAATCAGGAGGCGACCTGENCODING SEQ ID GTGCAGCCTGGCGGCTCACTGAGACTGAGCTGC NO: 12GCCGCTAGTGGCTTCACCTTTAGTAGCTACTGGA TGAGCTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAGTGGGTGGCCAATATTAAGCAGGACGGCTC AGAGAAGTACTACGTGGACTCAGTGAAGGGCCGGTTCACTATTAGCCGGGATAACGCTAAGAATAGC CTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCTAGAGATAGAGG CTCACTGTACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCT ALTERNATIVE PN 57 GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGAENCODING SEQ ID GCGCTAGTGTGGGCGATAGAGTGACTATCACCTG NO: 53TAGACCTAGTCAGGGGATTAACTGGGAGCTGGCC TGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCTAGTAGTCTGGAAAACG GCGTGCCCTCTAGGTTTAGCGGCTCAGGCTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTGCAG CCCGAGGACTTCGCTACCTACTACTGTCAGCAGTTTAACTCCTACCCCCTGACCTTCGGCGGAGGCAC TAAGGTGGAAATCAAG ALTERNATIVE PN 31GAGGTGCAGCTGGTGGAATCAGGAGGCGACCTG ENCODING SEQ IDGTGCAGCCTGGCGGCTCACTGAGACTGAGCTGC NO: 14GCCGCTAGTGGCTTCACCTTTAGTAGCTACTGGA TGAGCTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAGTGGGTGGCCAATATTAAGCAGGACGGCTC AGAGAAGTACTACGTGGACTCAGTGAAGGGCCGGTTCACTATTAGCCGGGATAACGCTAAGAATAGC CTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCTAGAGATAGAGG CTCACTGTACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCTGCTAGCACCAAGGGCCCAAGTG TCTTTCCCCTGGCCCCCAGCAGCAAGTCCACAAGCGGAGGCACTGCAGCTCTGGGTTGTCTGGTGAA GGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACC TTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTCGTGACTGTGCCTAGTTCCA GCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGA GTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGA GGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGG TGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGG GGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGT GTCCGTCCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACA AGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGT GTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAG GGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAG ACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGT CCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACAC CCAGAAGTCCCTGAGCCTGAGCCCCGGCAAGALTERNATIVE PN 58 GCTATTCAGCTGACTCAGTCACCTAGTAGCCTGA ENCODING SEQ IDGCGCTAGTGTGGGCGATAGAGTGACTATCACCTG NO: 54TAGACCTAGTCAGGGGATTAACTGGGAGCTGGCC TGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCTAGTAGTCTGGAAAACG GCGTGCCCTCTAGGTTTAGCGGCTCAGGCTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTGCAG CCCGAGGACTTCGCTACCTACTACTGTCAGCAGTTTAACTCCTACCCCCTGACCTTCGGCGGAGGCAC TAAGGTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGC TGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCA GTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAA GGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGT ACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGT GC LEADER SEQUENCE 59MEFGLSWVFLVAILEGVHC OF THE HEAVY CHAIN LEADER SEQUENCE 60MDMRVPAQLLGLLLLWLPGARC OF THE LIGHT CHAIN PN ENCODING SEQ 61ATGGAATTCGGCCTGAGCTGGGTGTTCCTGGTCG ID NO: 59 CGATTCTGGAAGGCGTGCACTGCPN ENCODING SEQ 62 ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGG ID NO: 60CTTCTGCTGCTCTGGCTCCCAGGCGCCAGATGT ALTERNATIVE 63 MAWVWTLPFLMAAAQSVQALEADER SEQUENCE OF THE HEAVY CHAIN ALTERNATIVE 64 MSVLTQVLALLLLWLTGTRCLEADER SEQUENCE OF THE LIGHT CHAIN ALTERNATIVE PN 65ATGGCCTGGGTGTGGACCCTGCCCTTCCTGATGG ENCODING SEQ IDCCGCTGCTCAGTCAGTGCAGGCC NO: 63 ALTERNATIVE PN 66ATGAGCGTGCTGACTCAGGTGCTGGCCCTGCTGC ENCODING SEQ IDTGCTGTGGCTGACCGGCACCCGCTGC NO: 64 SECOND 67 MEWSWVFLFFLSVTTGVHSALTERNATIVE LEADER SEQUENCE OF THE HEAVY CHAIN SECOND 68MSVPTQVLGLLLLWLTDARC ALTERNATIVE LEADER SEQUENCE OF THE LIGHT CHAINSECOND 69 ATGGAATGGTCCTGGGTGTTCCTGTTCTTCCTGTC ALTERNATIVE PNCGTGACCACAGGCGTGCACTCC ENCODING SEQ ID NO: 67 SECOND 70ATGTCCGTGCCCACACAGGTGCTGGGCCTGCTG ALTERNATIVE PNCTGCTGTGGCTGACCGACGCCAGATGC ENCODING SEQ ID NO: 68 CONSENSUS, 71SQX₁IX₂X₃X₄ CDRL1 (CHOTHIA) CONSENSUS, 72 FX₁SYPL CDRL3 (CHOTHIA)CONSENSUS, 73 RPSQX₁IX₂X₃X₄LA CDRL1 (KABAT) CONSENSUS, 74 DASSLEX₁CDRL2 (KABAT) CONSENSUS, 75 QQFX₁SYPLT CDRL3 (KABAT) huIL-17A 76GITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTN PKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCP NSFRLEKILVSVGCTCVTPIVHHVAEFRHhuIL-17F 77 MRKIPKVGHTFFQKPESCPPVPGGSMKLDIGIINENQRVSMSRNIESRSTSPWNYTVTWDPNRYPSEVVQ AQCRNLGCINAQGKEDISMNSVPIQQETLVVRRKHQGCSVSFQLEKVLVTVGCTCVTPVIHHVQ alternative 78GPIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNIHNR huIL-17ANTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVI WEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA cynoIL-17A 79GIAIPRNSGCPNSEDKNFPRTVMVNLNIHNRNTSTN PKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCVKADGNVDYHMNSVPIQQEILVLRREPRHC PNSFRLEKILVSVGCTCVTPIVHHVAcynoIL-17F 80 MRKIPKVGHTFFQKPESCPPVPEGSMKLDTGIINENQRVSMSRNIESRSTSPWNYTVTWDPNRYPSEVVQ AQCKHLGCINAQGKEDISMNSVPIQQETLVLRRKHQGCSVSFQLEKVLVTVGCTCVTPVVHHVQ rhesusIL-17A 81GIAIPRNSGCPNSEDKNFPRTVMVNLNIHNRNTSTS PKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCVKADGNVDYHMNSVPIQQEILVLRREPRHC PNSFRLEKILVSVGCTCVTPIVHHVAmarmosetIL-17A 82 SPQNPGCPNAEDKNFPRTVMVNLNIRNRNTNSKRASDYYNRSSSPWNLHRNEDPERYPSVIWEAKCRHLG CVDADGNVDYHMNSVPIQQEILVLRREPRHCTNSFRLEKMLVSVGCTCVTPIVHHVA mIL-17A 83 MAAIIPQSSACPNTEAKDFLQNVKVNLKVFNSLGAKVSSRRPSDYLNRSTSPWTLHRNEDPDRYPSVIWEA QCRHQRCVNAEGKLDHHMNSVLIQQEILVLKREPESCPFTFRVEKMLVGVGCTCVASIVRQAA mIL-17F 84APEPEFRHRKNPKAGVPALQKAGNCPPLEDNTVRV DIRIFNQNQGISVPREFQNRSSSPWDYNITRDPHRFPSEIAEAQCRHSGCINAQGQEDSTMNSVAIQQEILV LRREPQGCSNSFRLEKMLLKVGCTCVKPIVHQAAratIL-17A 85 MAVLIPQSSVCPNAEANNFLQNVKVNLKVINSLSSKASSRRPSDYLNRSTSPWTLSRNEDPDRYPSVIWEA QCRHQRCVNAEGKLDHHMNSVLIQQEILVLKREPEKCPFTFRVEKMLVGVGCTCVSSIVRHAS huIL-17RA 86NCTVKNSTCLDDSWIHPRNLTPSSPKDLQIQLHFAHTQQGDLFPVAHIEWTLQTDASILYLEGAELSVLQLNTNERLCVRFEFLSKLRHHHRRWRFTFSHF\NDPDQE YEVTVHHLPKPIPDGDPNHQSKNFLVPDCEHARMKVTTPCMSSGSLWDPNITVETLEAHQLRVSFTLWNESTHYQILLTSFPHMENHSCFEHMHHIPAPRPEEFHQRSNVTLTLRNLKGCCRHQVQIQPFFSSCLNDCLRHSA TVSCPEMPDTPEPIPDYMPLWEFRHDSGGGLNDIFEAQKIEWHE

1. A method of treating neuroinflammatory disease in a subject in needthereof comprising, administering to said subject an effective amount ofan IL-17A binding antibody or antigen-binding portion thereof, whereinsaid IL-17A binding antibody or antigen-binding portion thereofcomprises a heavy chain variable region (V_(H)) and a light chainvariable region (V_(L)); a. wherein said V_(H) comprises the threeComplementarity Determining Regions (CDRs) of the amino acid sequenceset forth as SEQ ID NO:12 and wherein said V_(L) comprises the threeCDRs of the amino acid sequence set forth as SEQ ID NO:43; b. whereinsaid V_(H) comprises, in sequence, the three CDRs set forth as aminoacid sequences SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:3 and wherein saidV_(L) comprises, in sequence, the three CDRs set forth as amino acidsequences SEQ ID NO:42, SEQ ID NO:23, SEQ ID NO:11; or c. c. whereinsaid V_(H) comprises, in sequence, the three CDRs set forth as aminoacid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and wherein saidV_(L) comprises, in sequence, the three CDRs set forth as amino acidsequences SEQ ID NO:41, SEQ ID NO:5, SEQ ID NO:6, and wherein saidantibody inhibits proinflammatory cytokine release from astrocytes. 2.The method according to claim 1, wherein the neuroinflammatory diseaseis selected from the group consisting of psychiatric, neurological andneurodegenerative disorders.
 3. The method according to claim 1, whereinsaid proinflammatory cytokine is selected from the group consisting ofIL-6, CXCL1, IL-8, GM-CSF, and CCL2; a. A method for inhibitingangiogenesis in a subject in need thereof comprising, administering tosaid subject an effective amount of an IL-17A binding antibody orantigen-binding portion thereof, wherein said L-17A binding antibody orantigen-binding portion thereof comprises a heavy chain variable region(V_(H)) and a light chain variable region (V_(L)); a. wherein said V_(H)comprises the three Complementarity Determining Regions (CDRs) of theamino acid sequence set forth as SEQ ID NO:12 and wherein said V_(L)comprises the three CDRs of the amino acid sequence set forth as SEQ IDNO:43; b. wherein said V_(H) comprises, in sequence, the three CDRs setforth as amino acid sequences SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:3 andwherein said V_(L) comprises, in sequence, the three CDRs set forth asamino acid sequences SEQ ID NO:42, SEQ ID NO:23, SEQ ID NO:11; or c.wherein said V_(H) comprises, in sequence, the three CDRs set forth asamino acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and whereinsaid V_(L) comprises, in sequence, the three CDRs set forth as aminoacid sequences SEQ ID NO:41, SEQ ID NO:5, SEQ ID NO:6, and wherein saidantibody inhibits angiogenesis.
 4. The method according to claim 1,wherein the antibody or antigen-binding portion thereof is a humanantibody or antigen-binding portion thereof, a monoclonal antibody orantigen-binding portion thereof, a chimeric antibody or antigen-bindingportion thereof, a humanized antibody or antigen-binding portionthereof, a F(ab′)2 fragment, a dAb fragment, a Fab fragment, or a Fvfragment.
 5. The method according to claim 4, wherein the antibody orantigen-binding portion thereof is a monoclonal antibody, a chimericantibody, or a humanized antibody.
 6. The method according to claim 4,wherein the antibody or antigen-binding portion thereof is a humanantibody.
 7. The method according to claim 6, wherein the human antibodycomprises a heavy chain and a light chain, wherein said heavy chaincomprises the amino acid sequence set forth as SEQ ID NO:14 and whereinsaid light chain comprises the amino acid sequence set forth as SEQ IDNO:44.
 8. The method according to claim 4, wherein the antibody orantigen-binding portion thereof is a human antibody or antigen-bindingportion thereof, a monoclonal antibody or antigen-binding portionthereof, a chimeric antibody or antigen-binding portion thereof, ahumanized antibody or antigen-binding portion thereof, a F(ab′)2fragment, a dAb fragment, a Fab fragment, or a Fv fragment.
 9. Themethod according to claim 8, wherein the antibody or antigen-bindingportion thereof is a monoclonal antibody, a chimeric antibody, or ahumanized antibody.
 10. The method according to claim 9, wherein theantibody or antigen-binding portion thereof is a human antibody.
 11. Themethod according to claim 10, wherein the human antibody comprises aheavy chain and a light chain, wherein said heavy chain comprises theamino acid sequence set forth as SEQ ID NO:14 and wherein said lightchain comprises the amino acid sequence set forth as SEQ ID NO:44.